KR20240074847A - Methods and systems for improving therapeutic cells - Google Patents

Abstract

The present invention relates to methods and systems for enriching cell populations (e.g., chondrocytes) for tissue engineering applications (e.g., production of new cartilage), wherein the methods and systems of the present invention provide It is characterized by the introduction of a storage solution, which results in new tissue (e.g., new cartilage) constructs being mechanically significantly more robust compared to those not treated with a storage solution. In addition, the method and system may further include a process of introducing cytochalasin D into the cells purified by the stock solution, which can further enhance the mechanical properties and matrix deposition of the cells. The methods and systems have the effect of enabling neocartilage engineered from chondrocytes, for example from fetal age tissue, to have compression properties equivalent to those of native adult articular cartilage.

Description

Methods and systems for improving therapeutic cells

The present invention relates to cell purification methods used in applications such as cell and tissue engineering as well as cell and tissue transplantation.

This application claims priority from U.S. Patent Application No. 17/496,391, filed October 7, 2021, the entire specification of which is incorporated herein by reference.

The goal of tissue engineering is to prevent and reverse disease progression by replacing damaged tissue. In general, fully differentiated cells are widely considered to be the ideal cell type for tissue engineering. They are phenotypically stable, readily produce tissue-specific extracellular matrix (ECM) molecules, and are most desirable in juvenile and fetal tissues, which have enhanced proliferative and synthetic abilities compared to adult cells. Tissue engineering products composed of juvenile cells are currently in clinical use. For example, RevaFlex (ISTO Technologies), a tissue engineering product for cartilage restoration using childhood chondrocytes, is currently undergoing phase 3 clinical trials in the United States. Although these engineered tissues show potential, they have not yet been able to reproduce the characteristics and structure of native cells.

In childhood and the fetus, fully differentiated cells are generally viewed as the ideal cell type for tissue engineering applications. However, their use in tissue engineering is difficult due to contamination with undesirable cell types, which prevents juvenile and fetal cells from achieving functional properties similar to those produced by adult-level cells or normal cells, and fetal-age chondrocytes. Increasing the mechanical properties of neocartilage from birth to adult levels has never been achieved previously.

Tissue engineering efforts using primary cells can be hindered by contamination by unwanted cell types. Contamination by blood and surrounding tissue may occur during separation of target donor tissue. Additionally, many cellular tissues are composed of various types of cells, and not all of these cells are suitable for tissue engineering applications. Disease states and tissue maturity can introduce additional undesirable cell phenotypes into isolated cell populations. Aging tissue, which is more susceptible to diseases such as cancer, arteriosclerosis and osteoarthritis, contains senescent cells that increasingly produce reactive oxygen species, inflammatory mediators and matrix-degrading enzymes. These limitations have required purification of cells during the isolation process to eliminate the presence of undesirable phenotypes (e.g., undesirable cytoskeletal properties) and to obtain a homogeneous cell population enriched with cells with properties suitable for tissue engineering. .

This invention was made with government support under grant number R01 AR067821 awarded by the NIH, and the government has certain rights in the invention.

The present invention provides methods and systems for improving cells for therapy. For example, it relates to a cell purification method for enriching cell populations by concentrating cell populations with properties advantageous for cell and tissue engineering.

Articular cartilage tissue engineering is well established and can be used as an example system. However, what is not generally recognized is that unwanted cell phenotypes in chondrocytes can exist for a variety of reasons. When examining cartilage biopsies in clinical applications such as autologous chondrocyte implantation (ACI), contamination by hematopoietic cells (e.g., proapoptotic cells) or cells from other surrounding tissues may occur. Short-term exposure of cartilage to blood has been shown to induce chondrocyte death in a model reflecting hemophilia. Second, in clinical settings, autologous or allogeneic cartilage grafts are often harvested from adult tissue that exhibits matrix degradation, surface defects, and fibrosis, and cartilage in diseases such as osteoarthritis exhibits increased extracellular matrix (ECM) degeneration and an altered phenotype. Contains cartilage cells. Degenerative changes in cartilage extracellular matrix (ECM) are associated with chondrocyte death. Fetal cartilage, on the other hand, is vascularized, allowing blood and various cell types to enter the tissue mass from which the cartilage cells are separated. In addition, even in normal tissue, cartilage separation itself causes damage to cellular tissue, causing necrosis at the edge of the wound and spreading the death wave into the cellular tissue. In addition to red blood cell (RBC) contamination, heterogeneity of cell phenotype caused by chondrocytes with altered phenotypes is a factor that unexpectedly limits the ability of engineered cartilage to reach the properties of native tissue.

Despite the potential for contamination during chondrocyte isolation, there have been few studies demonstrating its importance. As a result of dividing hamster rib cartilage into two fractions and sequentially digesting them with collagenase, it was confirmed that the second fraction contained a cell population with a more uniform chondrocyte morphology compared to the unseparated portion. . Another method to purify isolated chondrocytes is through sequential plate culture. Rat chondrocyte isolates isolated by differential attachment to tissue culture plastic showed 100% chondrocytes after the eighth plating, in contrast to the cell mixture when the entire population was plated. Another method is to use cell surface markers such as CD14 and CD45 to exclude contamination by monocytes and hematopoietic cells. Ammonium chloride-potassium chloride lysis buffer (ACK buffer) is commonly used to lyse red blood cells in samples containing white blood cells, such as EDTA-treated whole blood, yellow blood, and bone marrow. For tissue engineering purposes, ACK buffer is used to isolate pure stem cell populations, such as adipose-derived and mesenchymal stem cells, but has not yet been explored for the isolation of non-stem cell types (e.g., cartilage). Because fully differentiated cell isolates may contain cells with alternative phenotypes to contaminating cell types, ACK buffer treatment holds the potential for purification of cell populations desirable for tissue engineering applications. Although ACK buffer treatment has the potential to lyse all cells, the present invention preferentially destroys cells with a modified phenotype (e.g., cells in the early stages of apoptosis) and forms cells with a phenotype favorable for the formation of new cell tissue. make it possible

One of the unique and innovative technical features of the present invention is the treatment of freshly isolated, fully differentiated cells using a stock solution (e.g., ACK buffer) to remove early apoptotic cells and improve their ability to form viable cell tissue. It is ordered. It is not intended to limit the present invention to any particular theory or mechanism, and the methods and systems of the present invention are intended to improve the mechanical properties of neonatal tissue formed from specific cell populations (e.g., fetal age cells, diseased tissue sources) at adult levels. It appears that it can be improved to the same level as tissue made from cells or normal cells.

Additionally, the prior art deviates from the present invention. For example, the prior art utilizes hypotonic solution treatment for cells derived from fetal sheep or juvenile cattle, while the use of hypotonic solution treatment for cells derived from humans to remove early apoptotic cells is not discussed. Not mentioned. Figure 18 shows that treatments that are beneficial to cells of one species (e.g., fetal sheep) are not necessarily beneficial when applied to cells of another species (e.g., humans). Therefore, it is not obvious that the same treatment (e.g. stock solution) will act similarly on cells of different species.

Figure 19 also shows that even within the same species (e.g., juvenile Yucatan minipig), different cell types can respond differently to the same treatment or culture regimen. Therefore, treatments or culture regimens that are beneficial to one cell type (e.g., articular chondrocytes) may not achieve the same beneficial effect when applied directly to another cell type (e.g., rib chondrocytes).

Since the prior art describes storage solution treatment as being used on cell populations including blood cells, it is surprising that cells isolated from non-vascular tissue such as cartilage respond to ACK buffer treatment.

Additionally, while chondrocytes, which do not contain blood cells, respond to ACK buffer treatment in an unexpected manner by forming engineered new cartilage, prior art directs the process of ACK buffer treatment to stem cells.

It is surprising that treatment of chondrocytes with a stock solution such as ACK buffer, which previously selects for cells with undesirable cytoskeletal properties, unfavorable membrane properties, and altered stiffness, resulted in an enriched cell population.

Additionally, it was surprising that young normal cartilage contained cells with undesirable properties, such as inducing apoptosis, to the extent that the formation of engineered new cartilage was affected by the presence of these cells.

It has surprisingly been discovered that the methods and systems of the present invention generate engineered scaffold-free neocartilage from enhanced fully differentiated cells obtained from the treatments described herein, achieving compressive properties comparable to native adult articular cartilage, which have previously been described. An increase in the mechanical properties of neocartilage from fetal-age chondrocytes to adult levels has never been achieved. In addition, the present invention provides a method for concentrating a cell population suitable for developing new cartilage and further provides a method for improving cells for treatment by manipulating the cytoskeleton. For example, when a stock solution was used during chondrocyte purification, the homogeneity, matrix deposition, and mechanical properties of the new cartilage construct were significantly improved. The combination of hypotonic fluid and cytochalasin D (cytochalasin D) resulted in neocartilage engineered from fetal-age chondrocytes achieving compressive properties comparable to native adult articular cartilage. It is not intended to limit the present invention to a specific theory or mechanism, but in addition to reducing RBC contamination, chondrocytes of a modified phenotype, removing cellular obstructions to the self-assembly process, and removing apoptotic stimuli can improve new cartilage homogeneity within the new cell tissue. , it appears that chondrocyte distribution and extracellular matrix (ECM) deposition are improved, improving the biochemical and mechanical properties of the engineered tissue formed from treated cells.

These results are surprising because this level of mechanical strength has never been previously observed from fetal chondrocytes.

Additionally, the invention provides preparation of cells, methods of preparing cell populations, and methods of enriching cell populations for treatment. The present invention also provides methods of preparing tissue and methods of strengthening tissue for treatment.

The present invention provides methods for enriching chondrocyte samples (e.g., human chondrocyte samples). This method may include collecting a chondrocyte sample from cartilage cell tissue, and treating the chondrocyte sample with a storage solution (eg, ammonium chloride-potassium lysis buffer (ACK buffer)). In some embodiments, the method may be repeated multiple times alone or in combination with other treatments.

In some embodiments of the invention, methods of preparing chondrocyte samples (e.g., human chondrocyte samples) are provided. These methods may include collecting a chondrocyte sample (e.g., a human chondrocyte sample), wherein the chondrocyte sample (e.g., a human chondrocyte sample) is a mixed population of non-apoptotic chondrocytes and early apoptotic chondrocytes. Includes. It may also include processing the chondrocyte sample (e.g., treatment with a storage solution (e.g., ACK)). In some embodiments, the method may be repeated multiple times alone or in combination with other treatments.

Additionally, the present invention provides a method for preparing human chondrocyte samples. The method may include collecting a human chondrocyte sample derived from a portion of the rib and treating the human chondrocyte sample with a storage solution (eg, ammonium chloride-potassium lysis buffer (ACK buffer)).

The invention also provides production by a method comprising (a) collecting a cartilage sample and digesting the cartilage sample to obtain a cell suspension, and (b) treating the cell suspension with a stock solution to obtain a processed cell sample. Samples of processed cells may be provided. In some embodiments, the treated cell sample is suitable for production of new cartilage.

The present invention provides a process for 1) harvesting a population of somatic cells, 2) modifying the population of somatic cells with pre-existing undesirable properties (e.g., pre-existing undesirable cytoskeletal properties, pre-existing undesirable membrane surface area properties, or pre-existing undesirable rigidity properties). ), 3) a process of isolating and removing cells with pre-existing undesirable characteristics, and 4) pre-existing undesirable characteristics (e.g., pre-existing undesirable cytoskeletal characteristics). , provides a method of enriching a cell population that involves isolating the remaining cell population enriched with cells without pre-existing undesirable membrane surface area properties or pre-existing modified rigidity properties. The above steps may be repeated several times alone or in combination with other treatments.

Any feature or combination of features described herein is included within the scope of the present invention unless mutually contradictory as is apparent from the context, this specification, and the knowledge of a person skilled in the art. Additional advantages and aspects of the invention will become apparent from the following detailed description and claims.

The features and advantages of the present invention will become apparent by considering the following detailed description taken in conjunction with the accompanying drawings.
Figure 1 shows the pellet morphology, viability, and red blood cell (RBC) content of fetal sheep articular chondrocytes and juvenile bovine articular chondrocytes before and after ACK treatment. This figure shows that ACK treatment resulted in a change in cell pellet color and a significant decrease in RBC content.
Figures 2A-2H are diagrams showing the macroscopic morphology of new cartilage and selected parameters. Figure 2A shows that ACK treatment removed extensive spherical areas (indicated by white arrows) in fetal ovine articular chondrocyte neocartilage. Figure 2B shows that ACK treatment reduced the thickness of fetal sheep new cartilage. Figure 2C shows that ACK treatment reduced the water content of fetal sheep new cartilage. Figure 2D shows that ACK treatment did not affect hydration in fetal sheep. Figure 2E shows that ACK treatment removed extensive spherical areas (indicated by white arrows) in juvenile bovine articular chondrocyte neocartilage. Figure 2F shows that ACK treatment reduced the thickness of juvenile bovine neocartilage. Figure 2G shows that ACK treatment reduced the functional weight of juvenile bovine neocartilage. Figure 2H shows that ACK treatment did not affect hydration in juvenile cows.
Figure 3 is a diagram showing new cartilage histologically. ACK treatment of fetal sheep and juvenile bovine articular chondrocytes removes extensive areas of low cell density (*) present in untreated constructs, improves the homogeneity of new cartilage, and increases the concentration of glycosaminoglycans (GAGs), total collagen, and Collagen II staining was enhanced.
Figures 4A-4J are diagrams showing the biochemical content of new cartilage in fetal sheep articular chondrocytes (foACs) and juvenile bovine articular chondrocytes (jbACs) with or without ACK treatment. Figure 4A shows that ACK treatment significantly reduced caspase activity in foACs. Figure 4B shows that ACK treatment did not affect the glycosaminoglycan (GAG)/water weight content in foACs. Figure 4C shows that ACK treatment did not affect glycosaminoglycan (GAG)/dry weight (DW) content in foACs. Figure 4D shows that ACK treatment significantly increased collagen/water weight content in foACs. Figure 4E shows that ACK treatment significantly increased collagen/dry weight (DW) content in foACs. Figure 4F shows that ACK treatment significantly reduced caspase activity in jbACs. Figure 4G shows that ACK treatment significantly reduced the glycosaminoglycan (GAG)/hydrocytic weight content in jbACs. Figure 4H shows that ACK treatment significantly reduced glycosaminoglycan (GAG)/dry weight (DW) content in jbACs. Figure 4I shows that ACK treatment significantly increased collagen/water weight content in jbACs. Figure 4J shows that ACK treatment did not affect glycosaminoglycan (GAG)/water weight content in jbACs.
Figure 5 is a diagram showing the mechanical properties of new cartilage. ACK treatment significantly increased all measured mechanical properties in both cell types.
Figures 6A-6H are diagrams showing the effect of seeding density on the macroscopic morphology, biochemical content, and cell histology of new cartilage. Figure 6A shows macroscopic abnormalities at seeding densities of 5 and 4 million cells at P0 and P3R passages, respectively. Figures 6B and 6D show that glycosaminoglycan (GAG)/DNA (Figure 6B) and collagen/DNA (Figure 6D) of P3R new cartilage show a seeding density-dependent effect and are higher than those of P0 new cartilage. Figure 6F shows that the pyridinoline content of P0 new cartilage is higher than that of P3R new cartilage. Figures 6C, 6E, and 6G show that mechanical properties such as aggregate modulus (Figure 6C), tensile modulus (Figure 6E), and ultimate tensile strength (Figure 6G) increase with seeding density of P0 cells and decrease with seeding density of P3R cells. It shows that Figure 6H shows H&E staining and immunohistochemistry (IHC) staining for glycosaminoglycan (GAG), collagen type I (col I), collagen type II (col II), and total collagen (total col). IHC controls are meniscus (M), articular cartilage (AC), and tendon (T) (stage 1).
Figure 7 is a diagram showing phenotypic verification of engineered new cartilage. At this time, histological controls are articular cartilage (AC) and growth plate (GP).
Figures 8A-8H are diagrams showing the effect of cytochalasin D (cytochalasin D; Cyto D) and hyaluronidase (Hya) treatment on P3R new cartilage. Figure 8A shows that macroscopic abnormalities appeared only in the Hya treatment group. Figures 8C, 8D, 8F, and 8H show the glycosaminoglycan (GAG)/water weight (Figure 8C) and the mechanical properties of aggregate modulus (Figure 8D), tensile modulus (Figure 8F), and ultimate tensile strength (Figure 8H). It shows an increase due to Cyto D treatment. Figures 8E and 8G show that collagen (Figure 8E) and pyridinoline (Figure 8G) content did not change with any treatment. Figure 8B shows H&E staining and IHC staining for glycosaminoglycan (GAG), collagen type I (col I), collagen type II (col II), and total collagen (total col). IHC controls are meniscus (M), articular cartilage (AC), and tendon (T) (stage 2).
Figure 9 is a diagram showing the effect of cytochalasin D (cytochalasin D) treatment on actin arrangement. Cytochalasin D treatment improved the cortical arrangement of actin in both P3 and P3R chondrocytes (step 2).
Figures 10A-10H show the effect of cytochalasin D (Cyto D) and TCL treatment on P3R new cartilage. Figure 10A shows that there are no macroscopic abnormalities. Figure 10B shows H&E staining and IHC staining for glycosaminoglycan (GAG), collagen type I (col I), collagen type II (col II), and total collagen (total col). IHC controls are meniscus (M), articular cartilage (AC), and tendon (T). Figures 10E and 10G show that TCL treatment in combination with Cyto D (Cyto D + TCL) increased collagen (Figure 10E) and pyridinoline (Figure 10G) content and glycosaminoglycan (GAG) content (Figure 10C). ) shows that no significant difference was observed. Figures 10F and 10H show that TCL treatment in combination with Cyto D (Cyto D + TCL) increased tensile stiffness (Figure 10F) and strength (Figure 10H) (step 3). No significant differences were observed in the aggregates (Figure 10D).
Figures 11A-11E are diagrams showing the increase in functional properties of new cartilage. Figure 11A shows a 9.6-fold increase in collective modulus. Figure 11B shows a 7.2-fold increase in shear modulus. Figure 11C shows a 3.8-fold increase in tensile modulus. Figure 11D shows a 9.0-fold increase in ultimate tensile strength. Figure 11E shows that P3R new cartilage exceeded fetal and juvenile native tissue values and approached adult levels (stages 1-3).
Figures 12A-12B are diagrams showing the effect of cytochalasin D (cytochalasin D; Cyto D) and hyaluronidase (Hya) treatment on P3 new cartilage. Figure 12A shows that Cyto D treatment resulted in uniquely flat structures. Figure 12B shows H&E staining and IHC staining for glycosaminoglycan (GAG), collagen type I (col I), collagen type II (col II), and total collagen (total col). IHC controls (B) are meniscus (M), articular cartilage (AC), and tendon (T) (stage 2).
Figure 13 is a diagram showing Table 1 (stage 1 data). Data were expressed as mean ± standard deviation, and statistics were calculated across groups within biochemical or mechanical parameters. Additionally, statistical significance is indicated for groups indicated by different letters.
Figure 14 is a diagram showing Table 2 (second stage data, P3). Data were expressed as mean ± standard deviation, and statistics were calculated across groups within biochemical or mechanical parameters. Additionally, statistical significance is indicated in groups indicated by different letters.
Figure 15 is a diagram showing Table 3 (step 2). Data were expressed as mean ± standard deviation, and statistics were calculated across groups within biochemical or mechanical parameters. Additionally, statistical significance is indicated for groups indicated by different letters.
Figure 16 is a diagram showing Table 4 (step 3). Data were expressed as mean ± standard deviation, and statistics were calculated across groups within biochemical or mechanical parameters. Additionally, statistical significance is indicated for groups indicated by different letters.
Figure 17 is a diagram summarizing compression characteristics. The aggregate elastic modulus of ACK buffer-treated P3R cells seeded at optimal density increased 9.6-fold compared to the P0 control group when treated with cytochalasin D (cytochalasin D).
Figure 18 shows that chondrocytes derived from fetal sheep articular cartilage or adult human costal cartilage were passaged three times (P3), underwent aggregate redifferentiation (rejuvenation), and seeded 2 million cells per new cartilage construct using a self-assembly process. This is a drawing that shows. The control construct was not treated, and the construct treated with cytochalasin D (Cyto D) was treated at 2 μM from the 01st to the 21st rounds. When Cyto D was applied to new cartilage constructs formed from fetal ovine articular chondrocytes (foACs), both compressive aggregate modulus and shear modulus were significantly increased, which is a beneficial effect. However, in contrast, when Cyto D was applied to adult human costal chondrocytes (ahCCs), the aggregate modulus and shear modulus were significantly reduced. The examples clearly show that treatments that are beneficial to cells of a particular species may not be beneficial when applied to cells of another species.
Figure 19 is a diagram showing that chondrocytes derived from juvenile Yucatan minipig articular cartilage (jyACs) or juvenile Yucatan minipig costal cartilage (jyCCs) were passaged three times (P3) and underwent aggregate redifferentiation (rejuvenation). In the control group, 2 million jyACs or jyCCs self-assembled to form a new cartilage structure. For the experimental group, 2 million jyACs or jyCCs initially underwent self-assembly of new cartilage, and then 2 million cells were additionally seeded on top 1, 2, 3, or 4 hours later, aiming to increase the thickness of the structure. New cartilage derived from jyACs with the addition of a second cell layer at 1, 2, 3, and 4 hours was significantly thicker than the control construct, and construct diameter was not affected. However, in the case of new cartilage derived from jyCCs, the thickness was not affected by the cell layer added at any specific time point, and the diameter of the structure significantly decreased with the addition of cells at all time points. This clearly shows that treatments or culture regimens that are beneficial to one cell type may not achieve beneficial effects when applied directly to other cell types, even within the same species.
Figure 20 is a diagram showing ACK-treated human costal chondrocytes from adult and childhood donors. Chondrocytes derived from adult or juvenile human costal cartilage were treated with stock solution (ACK buffer) according to the procedure described herein. Briefly, the entire cell population freshly isolated from rib tissue was subjected to ACK treatment for 10 min to enrich the non-apoptotic early cell population. The non-apoptotic early cell fraction then proliferated until the third passage (P3), underwent aggregate redifferentiation (rejuvenation), and self-assembled into 2 million cells per new cartilage construct. Results: ACK-treated human costochondrocytes derived from adult and juvenile donors each produced morphologically normal (i.e., flat, non-curled) neocartilage constructs. The new cartilage construct had a uniform thickness and contained phenotypically normal extracellular matrix and living chondrocytes, and the construct also showed mechanical properties for compressive strength (collective modulus) and tensile strength (tensile modulus).

Unless otherwise specified, all technical and scientific terms used in this specification have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains.

As used herein, singular expressions such as a, an, and the are intended to include plural forms as well, unless the context clearly dictates otherwise. Additionally, the description and/or claims include “including,” “includes,” “having,” “has,” “with,” or variations thereof. To the extent used, these terms are intended to be inclusive in a manner similar to the term “comprising.”

As used herein, the term “apoptosis” refers to a form of programmed cell death that occurs in multicellular organisms. Biochemical reactions lead to characteristic cellular changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, DNA fragmentation, and mRNA reduction.

As used herein, the term “pre-apoptotic” refers to an altered state distinct from apoptosis. In some embodiments, initiating apoptosis cells share some characteristics with apoptotic cells, but initiating apoptosis is reversible, and apoptosis must be induced in addition to this process for cells to die. Characteristics of early apoptotic cells may include, but are not limited to, reduced cell membrane surface area, changes in cell rigidity (e.g., undesirable rigidity properties, see below), or cytoskeletal changes (e.g., undesirable cytoskeletal properties, see below). No. In some embodiments, initiating apoptosis and apoptosis are two distinct cellular states. In other embodiments, initiating apoptosis and apoptosis are two distinct cellular states.

As used herein, the term “pro-apoptotic” refers to a protein and/or cell that can cause another cell to enter an apoptotic or apoptotic state.

As used herein, the term "enhancement" refers to cell fractionation (enhancement of cell fraction ( This means generating a cell sample after processing.

As used herein, the term “undesirable cell type” may refer to cells that are unsuitable for tissue engineering applications, such as early-apoptotic cells or apoptosis-inducing cells (e.g., red blood cells).

As used herein, the term "undesirable cytoskeletal characteristics" means cells with a weakened, fragmented, disrupted or altered cytoskeleton, cells that are unable to reform or have a reduced ability to reform. refers to a cell with a skeleton, a cell with cytoskeletal characteristics that are susceptible to destruction by treatment (e.g., treatment with a storage solution such as ACK buffer), or a cell with a combination of the above characteristics.

As used herein, the term "undesirable membrane characteristics" refers to cells with reduced membrane surface area, cells with disrupted or deformed membranes, cells with membranes that are unable to adapt to structural changes/size changes, or cells with a membrane that is unable to adapt to structural changes/size changes, treatment (e.g. : refers to cells with membrane characteristics that are susceptible to destruction by treatment with a storage solution such as ACK buffer, or cells with a combination of the above characteristics.

As used herein, the term "undesirable stiffness characteristics" refers to cells with reduced overall stiffness, cells with increased overall stiffness, cells with different stiffness depending on the cell region examined, and cells with reduced flexibility. , refers to cells with rigidity characteristics that are susceptible to destruction by treatment (e.g., treatment with a storage solution such as ACK buffer) or cells with a combination of the above characteristics.

As used herein, the term “undesirable” refers to a property that has a deleterious effect on the cell. For example, cells with undesirable properties (including, but not limited to, undesirable cytoskeletal properties, undesirable membrane properties, undesirable rigidity properties, or combinations thereof) may respond appropriately to various stresses. Less likely and therefore more susceptible to extinction (e.g. harmful effects). Additionally, cells with undesirable properties may produce an extracellular matrix of altered composition (e.g., collagen I instead of collagen II), may produce less extracellular matrix overall, and may have altered mechanical properties, e.g. It can produce an extracellular matrix with reduced stiffness and strength.

As used herein, a "pre-existing" characteristic (e.g., characteristic) is a cell that is present during or after collection of a cell sample but is not present after processing the cell population (or cells) by the methods described herein. It refers to the characteristics of (or cell population).

As used herein, “chondrocytes” are the only cells found in normal cartilage and produce and maintain cartilage matrix, which may include collagen and proteoglycans.

As used herein, “cartilage” is a non-vascular type of supportive connective tissue found throughout the body. There are three types of cartilage: hyaline cartilage (non-articular cartilage, such as rib cartilage), fibrocartilage, and elastic cartilage.

Details are set forth herein to summarize certain aspects, advantages and novel features of the invention. It should be understood that not all such advantages will necessarily be achieved in accordance with particular embodiments of the invention. Accordingly, the invention may not necessarily achieve a single advantage or group of advantages as described herein, but may be implemented or performed in an optimal manner.

Additionally, although embodiments of the invention have been described in detail, certain variations and modifications will be apparent to those skilled in the art, including embodiments that do not provide all the features and advantages described herein. It will be understood by those skilled in the art that the invention extends beyond the specifically disclosed embodiments to other alternative or additional embodiments and/or uses and obvious modifications and equivalents thereof. Moreover, although various modifications have been shown and described in various details, other modifications within the scope of the present invention will be readily apparent to those skilled in the art based on the present invention. It is also believed that various combinations or sub-combinations of specific features and aspects of the embodiments can be made and still be included within the scope of the present invention. Accordingly, it should be understood that various features and aspects of the disclosed embodiments may be combined with or substituted for one another to obtain various forms of the present invention. Accordingly, the scope of the invention disclosed herein should not be limited by the specific embodiments described herein.

The present invention provides a method of preparing (e.g., enriching) a chondrocyte sample (e.g., a human chondrocyte sample). This method may include collecting a chondrocyte sample from cartilage tissue, and treating the chondrocyte sample with a storage solution (eg, ammonium chloride-potassium lysis buffer (ACK buffer)). In other embodiments, this method may be repeated multiple times alone or in combination with other treatments.

The invention may also provide methods for preparing (e.g., enriching) non-articular chondrocyte samples. This method may include collecting a non-articular chondrocyte sample from cartilage tissue, and treating the non-articular chondrocyte sample with a storage solution (eg, ammonium chloride-potassium lysis buffer). In other embodiments, this method may be repeated multiple times alone or in combination with other treatments.

The present invention provides a method of preparing (e.g., enriching) a chondrocyte sample (e.g., a human chondrocyte sample). These methods may include collecting a chondrocyte sample (e.g., a human chondrocyte sample) and processing the chondrocyte sample (e.g., a stock solution, such as ACK buffer). In another embodiment, the method includes collecting a chondrocyte sample (e.g., a human chondrocyte sample) and treating the chondrocyte sample with a stock solution (e.g., ACK). In some embodiments, a chondrocyte sample (e.g., a human chondrocyte sample) includes a mixed population of non-apoptotic chondrocytes and early apoptotic chondrocytes. In other embodiments, this method may be repeated multiple times alone or in combination with other treatments.

In some embodiments, a post-treatment chondrocyte sample (e.g., a human chondrocyte sample) has a higher percentage of non-apoptotic cells compared to a pre-treatment chondrocyte sample. In some embodiments, a post-treatment chondrocyte sample (e.g., a human chondrocyte sample) has a lower percentage of cells in early stage of apoptosis compared to a pre-treatment chondrocyte sample. In some embodiments, a post-treatment chondrocyte sample (e.g., a human chondrocyte sample) has a lower percentage of apoptotic cells compared to a pre-treatment chondrocyte sample. In another embodiment, a post-treatment chondrocyte sample (e.g., a human chondrocyte sample) has a lower percentage of undesirable cell types compared to a pre-treatment chondrocyte sample.

Additionally, the present invention may provide methods for preparing (e.g., enriching) cell populations. These methods may include collecting a sample of cells from a portion of a rib and processing the sample of cells from a portion of a rib (e.g., a stock solution, such as ACK buffer). In another embodiment, the method includes taking a sample of cells from a portion of a rib and treating the sample of cells from a portion of the rib with a stock solution (e.g., ACK). In some embodiments, a cell sample derived from a portion of a rib includes a mixed population of non-apoptotic and early apoptotic cells. In other embodiments, this method may be repeated multiple times alone or in combination with other treatments.

In some embodiments, a sample of cells derived from a portion of a rib after treatment has a higher percentage of non-apoptotic cells compared to a sample of cells derived from a portion of a rib prior to treatment. In some embodiments, a sample of cells derived from a portion of a rib after treatment has a lower percentage of cells in early stage of apoptosis compared to a sample of cells derived from a portion of a rib prior to treatment. In some embodiments, a sample of cells derived from a portion of a rib after treatment has a lower percentage of apoptotic cells compared to a sample of cells derived from a portion of a rib prior to treatment. In another embodiment, a sample of cells derived from a portion of a rib after treatment has a lower percentage of undesirable cell types compared to a sample of cells derived from a portion of a rib prior to treatment.

The invention may also provide methods for preparing (e.g., enriching) non-articular chondrocyte samples. This method may include collecting a non-articular chondrocyte sample and processing the non-articular chondrocyte sample (e.g., a stock solution, such as ACK buffer). In another embodiment, the method includes collecting a non-articular chondrocyte sample and treating the non-articular chondrocyte sample with a reservoir fluid (e.g., ACK). In some embodiments, the non-articular chondrocyte sample includes a mixed population of non-apoptotic chondrocytes and early apoptotic chondrocytes. In some embodiments, this method may be repeated multiple times alone or in combination with other treatments.

In some embodiments, a non-articular chondrocyte sample after treatment has a higher percentage of non-apoptotic cells compared to a non-articular chondrocyte sample before treatment. In some embodiments, a non-articular chondrocyte sample after treatment has a lower percentage of cells in early stage of apoptosis compared to a non-articular chondrocyte sample before treatment. In some embodiments, a non-articular chondrocyte sample after treatment has a lower percentage of apoptotic cells compared to a non-articular chondrocyte sample before treatment. In another embodiment, a non-articular chondrocyte sample after treatment has a lower percentage of undesirable cell types compared to a non-articular chondrocyte sample before treatment.

The invention also provides methods for preparing (e.g., enriching) human cell populations. These methods may include collecting a human cell sample from a portion of the rib and processing the human cell sample (e.g., in a stock solution, such as ACK buffer). In another embodiment, the method includes taking a human cell sample from a portion of the rib and treating the cell sample with a stock solution (e.g., ACK). In other embodiments, this method may be repeated multiple times alone or in combination with other treatments. In some embodiments, a cell sample derived from a portion of a rib includes a mixed population of non-apoptotic and early apoptotic cells. In other embodiments, this method may be repeated multiple times alone or in combination with other treatments.

In some embodiments, the methods described herein further include passage of cells in a monolayer or three-dimensional environment following treatment. In another embodiment, the methods described herein further include producing new cartilage from the passaged cells.

In some embodiments, the sample includes chondrocytes. In another embodiment, the sample includes human chondrocytes. In some embodiments, the sample includes non-articular chondrocytes. In another embodiment, the sample includes human non-articular chondrocytes. In some embodiments, the sample may be derived from cells derived from cartilage, such as any cartilage, including but not limited to hyaline cartilage, fibrocartilage, and elastic cartilage. In some embodiments, the sample includes cells derived from a portion of a rib. In another embodiment, the sample includes cells derived from a portion of a human rib. In some embodiments, a portion of a rib includes rib chondrocytes. In another embodiment, a portion of a rib includes rib tissue. In a further embodiment, a portion of a rib includes rib chondrocytes. In some embodiments, the rib tissue includes chondrocytes.

In some embodiments, the cell sample is a chondrocyte sample. In another embodiment, the cell sample is a non-articular chondrocyte sample. In some embodiments, the cell sample is a human cell. In another embodiment, the cell sample is from a portion of a rib. In some embodiments, the ribs include chondrocytes. In some embodiments, the chondrocytes are non-articular chondrocytes.

In some embodiments, the treatment includes adding a stock solution to the cell sample to induce cell expansion. Any stock solution, for example ammonium chloride-potassium lysis buffer (ACK buffer) or water, can be used according to the methods described herein. In some embodiments, the stock solution is ammonium chloride-potassium lysis buffer (ACK buffer). In another embodiment, processing includes adding a stock solution to the obtained cell sample. In some embodiments, the treatment induces expansion of cells. In some embodiments, the storage solution induces expansion of cells. In some embodiments, swelling of cells causes cell death. In some embodiments, the treatment preferentially induces expansion-related death of cells in early stages of apoptosis. In another embodiment, the storage solution preferentially induces expansion-related death of cells in early stages of apoptosis. In a further example, ACK buffer preferentially induces expansion of apoptotic cells.

It is not intended to limit the present invention to any particular theory or mechanism, and that cells in the early stages of apoptosis have altered cytoskeletal properties (e.g., undesirable cytoskeletal properties) and altered membrane properties (e.g., unfavorable membrane properties). They appear to be more sensitive to treatment (e.g. treatment with storage solutions (e.g. ACK buffer)) because the cells are more likely to burst. For example, if a treatment (e.g., treatment with a stock solution (e.g., ACK buffer)) is applied to a sample containing a mixed population of pro-apoptotic and non-apoptotic cells, the treatment will cause expansion of both cell populations. will lead you to do so. However, non-apoptotic cells can successfully reorganize their membranes and cytoskeleton to compensate for the increase in intracellular fluid, while apoptotic cells have difficulty reorganizing their membranes and cytoskeleton and are prone to bursting when treatments are applied. You can.

It is not intended to limit the present invention to any particular theory or mechanism, but when a treatment (e.g., mechanical treatment such as shear or compression) is applied to a sample containing a mixed population of apoptotic and non-apoptotic cells, the Cells in the early stages of apoptosis may be able to successfully reorganize their membranes and cytoskeleton to compensate for mechanical stress. However, cells in the early stages of apoptosis appear to be more vulnerable to applied treatments due to their difficulty in reorganizing their membranes and cytoskeleton.

In some embodiments, a treatment (e.g., a treatment comprising a stock solution such as ACK buffer) reduces early apoptotic cells in a sample. In some embodiments, a treatment (e.g., a treatment comprising a stock solution such as ACK buffer) removes a portion of early apoptotic cells from the sample. In another embodiment, a treatment (e.g., a treatment comprising a stock solution, e.g., ACK buffer) reduces apoptosis-inducing cells in a sample. In some embodiments, a treatment (e.g., a treatment comprising a stock solution such as ACK buffer) removes a portion of the apoptotic cells from the sample.

In some embodiments, post-processed cell samples (e.g., chondrocytes, human chondrocytes, non-articular chondrocytes, cells derived from a portion of a rib, etc.) may be used for one or more of the following: direct use of the cells, suspension culture, etc. In vitro culture of cells, including passaging in a monolayer or three-dimensional environment, tissue engineering, cell transplantation, tissue transplantation and/or implantation using non-scaffold systems, including self-assembly, or scaffold-based systems, including natural and synthetic materials.

In another embodiment, the processed cell sample (e.g., chondrocytes, human chondrocytes, non-articular chondrocytes, cells derived from a portion of a rib, etc.) or tissue engineered/fabricated from the cell sample produced above has one or more of the following: Additional processing may include: growth factors, cytoskeletal regulators, hormones, toxic compounds, molecules acting upstream in signaling pathways, various oxygen tensions, cross-linking agents, matrix degrading enzymes, matrix molecules and/or mechanical agents. Stimulation.

In some embodiments, the method of preparing a cell population further comprises culturing the population of non-apoptotic cells to produce new cartilage. In another embodiment, the method of preparing a population of human cells further includes culturing the population of non-apoptotic cells for production of new cartilage. In some embodiments, the methods described herein can be repeated multiple times, alone or in combination with other treatments.

The present invention provides methods and systems for improving cells for therapy, such as cell purification methods to enrich cell populations. Cells are used in tissue engineering applications and cell or cell tissue transplantation. The cell population may include fully differentiated cells such as chondrocytes, osteoblasts, adipocytes, and cardiomyocytes. Cellular tissue may include fat, cartilage, bone, tendons, ligaments, muscle, and skin.

Enhancement of cell populations improves tissue engineering by improving cell homogeneity with properties suitable for cell/tissue engineering, improving cell strength, improving cell phenotype, for example, leading to faster new tissue production or better new tissue constructs. It is considered an improvement in characteristics that leads to

The present invention provides a method of 1) isolating cells or tissue from, for example, a donor or source, and 2) treating cells (eg, chondrocytes) chemically or physically/mechanically. Non-limiting examples of chemical treatments include introducing a stock solution into cells during cell purification to produce significantly more mechanically robust new cell tissue constructs (e.g., new cartilage). This method may further include pelleting the cells.

The present invention provides purification methods based on cell properties, including cytoskeleton, membrane surface area, and rigidity properties. It is not intended to limit the present invention to a particular theory or mechanism, but the purification process preferentially selects cells with pre-existing undesirable characteristics or cells with an altered phenotype (damaged cells), which have a fragmented cytoskeleton, reduced including, but not limited to, increased membrane surface area and altered cell stiffness. These damaged cells are removed, creating a cell population enriched with functional cells and cells with characteristics favorable for the development of new tissue.

In some embodiments, the cells removed by treatment comprise more than 1% of the cartilage-derived cell or tissue population, and the cells removed have preexisting undesirable cytoskeletal, membrane surface area, and/or rigidity properties. It is designated as The cells or tissue populations used in accordance with the invention may be cells freshly extracted from cartilage of a living subject, cells previously frozen or otherwise preserved, or cells previously cultured in vitro or in vivo. there is.

In some embodiments, cells with pre-existing undesirable cytoskeletal properties include cells with a weakened, fragmented, disrupted, or altered cytoskeleton, cells that are unable to reform, or cells with a cytoskeleton that has a reduced ability to reform. , cells with cytoskeleton properties that are susceptible to destruction by treatment, or a combination thereof. Without intending to limit the present invention to any particular theory or mechanism, it appears that at least 1% of the cell population (e.g. chondrogenic cell population) has pre-existing undesirable cytoskeletal properties. Accordingly, in some embodiments, treatment using chemical or physical methods (e.g., expansion, shear, compression) may be used to remove cells with pre-existing undesirable cytoskeletal properties (e.g., chondrocytes) to remove cell populations (e.g., chondrocytes). Aim to remove at least 1% (but less than 99%) of the chondrogenic cell population. For example, screening conditions can be set to cause removal of at least 1% (but less than 99%) of the cell population based on pre-existing undesirable cytoskeletal properties. In some embodiments, treating at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or Removing at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75% aims to do so.

In some embodiments, cells with undesirable membrane properties include cells with reduced membrane surface area, cells with disrupted or deformed membranes, cells with membranes that cannot adapt to structural changes/size changes, and cells with membrane properties that are susceptible to disruption by treatment. cells or combinations thereof. Without wishing to limit the present invention to any particular theory or mechanism, it appears that at least 1% of the cell population (e.g. chondrogenic cell population) has pre-existing undesirable membrane surface properties. Accordingly, in some embodiments, treatment using chemical or physical methods (e.g., swelling, shearing, compression) may be used to remove cells with pre-existing undesirable membrane surface area properties (e.g., chondrocytes) to remove cell populations (e.g., chondrocytes). Aim to remove at least 1% (but less than 99%) of the chondrogenic cell population. For example, screening conditions can be set to cause removal of at least 1% (but less than 99%) of the cell population based on pre-existing undesirable membrane surface area properties. In some embodiments, treating at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or Removing at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75% aims to do so.

In some embodiments, cells with undesirable stiffness properties include cells with reduced overall stiffness, cells with increased overall stiffness, cells with different stiffness depending on the cell region examined, cells with reduced flexibility, and cells susceptible to destruction by treatment. Contains cells with easy rigidity properties or a combination thereof. Without intending to limit the present invention to any particular theory or mechanism, it appears that at least 1% of the cell population (e.g. chondrogenic cell population) has pre-existing undesirable stiffness properties. Accordingly, in some embodiments, treatment using chemical or physical methods (e.g., expansion, shear, compression) may be used to remove cell populations (e.g., cartilage) to remove cells (e.g., chondrocytes) with pre-existing undesirable stiffness properties. Aim to remove at least 1% (but less than 99%) of the forming cell population. For example, screening conditions can be set to cause removal of at least 1% (but less than 99%) of the cell population based on pre-existing undesirable stiffness properties. In some embodiments, treatment is performed on at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least of the cell population to remove cells with pre-existing undesirable rigidity properties. removing 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75% aims to

In appropriate circumstances, purification involves subjecting a population of cells to a treatment that 1) induces cell expansion, 2) induces shear, 3) applies shock or compression, or a combination thereof.

Non-limiting examples of methods for inducing cell expansion include adding a storage solution (e.g., ACK buffer) or water, performing freeze-thaw cycles, depressurizing dissolved gases, and applying vacuum or negative pressure. , or a combination of these.

Examples of methods to induce shear include, but are not limited to, shearing fluid flow, opposing microfluidic flow, forcing cells through small filters/mesh or pathways/tunnels, nebulizing solutions, or combinations thereof. No. As used herein, “small” filter/mesh refers to a filter or mesh that is less than 100 μm. In some embodiments, a “small” filter/mesh is about 10 μm to 100 μm, or about 10 μm to 90 μm, or about 10 μm to 80 μm, or about 10 μm to 70 μm, or about 10 μm to 60 μm. , or about 10 μm to 50 μm, or about 10 μm to 40 μm, or about 10 μm to 30 μm, or about 10 μm to 20 μm, or about 15 μm to 20 μm, or about 50 μm to 100 μm, or A filter or mesh of about 50 μm to 90 μm, or about 50 μm to 80 μm, or about 50 μm to 70 μm, or about 50 μm to 60 μm, or about 80 μm to 100 μm, or about 80 μm to 90 μm. means.

Non-limiting examples of methods of applying shock or inducing compression include forcing the material through a small filter/mesh or path/tunnel, applying mechanical compression, applying physical impact, or combinations thereof. In some embodiments, the purification method further includes subjecting the cells to high frequency vibration, such as sonication or cavitation generation. In some embodiments, “high frequency” means frequencies greater than 10 kHz. In some embodiments, the purification method is performed between about 10 kHz and 100 kHz, or between about 10 kHz and 90 kHz, or between about 10 kHz and 80 kHz, or between about 10 kHz and 70 kHz, or between about 10 kHz and 60 kHz, or about 10 kHz. kHz to 50 kHz, or about 10 kHz to about 45 kHz, or about 10 kHz to about 40 kHz, or about 10 kHz to 35 kHz, or about 10 kHz to 30 kHz, or about 10 kHz to 25 kHz, or about 10 kHz to 20 kHz, or about 10 kHz to 15 kHz, or about 20 kHz to 100 kHz, or about 20 kHz to 90 kHz, or about 20 kHz to 80 kHz, or about 20 kHz to 70 kHz, or about 20 kHz to 60 kHz kHz, or about 20 kHz to 50 kHz, or about 20 kHz to 45 kHz, or about 20 kHz to about 40 kHz, or about 20 kHz to 35 kHz, or about 20 kHz to 30 kHz, or about 20 kHz to 25 kHz , or about 50 kHz to 100 kHz, or about 50 kHz to 90 kHz, or about 50 kHz to 80 kHz, or about 50 kHz to 70 kHz, or about 50 kHz to 60 kHz, or about 80 kHz to 100 kHz, or It further includes treating the cells with vibration of about 80 kHz to 90 kHz. In other embodiments, the purification method is performed at about 10 kHz, or about 15 kHz, about 20 kHz, about 25 kHz, about 30 kHz, about 35 kHz, about 40 kHz, about 45 kHz, or about 50 kHz, or about 60 kHz. , or about 70 kHz, or about 80 kHz, or about 90 kHz, or about 100 kHz. In some embodiments, the purification method further includes subjecting the cells to vibration greater than 100 kHz.

In some embodiments, the stock solution includes ammonium chloride potassium (ACK) buffer. The ACK buffer may have a composition such as 154 mM ammonium chloride, 10 mM potassium bicarbonate, and 97 μM EDTA, but the ACK buffer is not limited to this composition. In appropriate circumstances, stock solutions include Gey's buffer, Tris-HCl, HEPES + EGTA + MgCl, MP-40 lysis buffer, RIPA lysis buffer, SDS, storage saline, diluted PBS, purified water, or combinations thereof. The present invention is not limited to the above-mentioned stock solutions.

The process of isolating cells from a donor or source involves collecting tissue from the donor and breaking down the tissue with enzymes including collagenase, dispase, pronase, or a combination thereof. It may include a process of filtering cells from enzyme-digested tissue, and resuspending cells in a buffer (e.g., stock solution or replacement buffer) or culture medium.

Any appropriate cell population can be used. For example, the cells can be mammalian cells or plant cells. In some embodiments, the cells include chondrocytes (e.g., primary chondrocytes), osteoblasts, cardiomyocytes, adipocytes, hepatocytes, tenocytes, osteoclasts, smooth muscle cells, pericytes, neurons, fibroblasts, and keratinocytes. , endothelial cells, muscle cells, mesenchymal stem cells, hematopoietic stem cells, adipose-derived stem cells, or a combination thereof. In some embodiments, a cell population is a combination of cell types. The present invention is not limited to the above-mentioned cell types or origins of the cells.

In some embodiments, the cells are normal cells. In some embodiments, the cells are from a diseased tissue or source (e.g., osteoarthritis cartilage).

The method of the present invention is a method of adding a cytoskeleton regulator, an actin polymerization inhibitor (e.g. cytochalasin D) and/or a cytoskeleton polymerization regulator ( It further includes the process of introducing an inhibitor or promoter, e.g. an inhibitor of polymerization of microtubules. Cytoskeleton modulators and/or actin polymerization inhibitors and/or cytoskeleton polymerization modulators may further enhance the mechanical properties and matrix deposition of the cell. The present invention is not limited to cytochalasin D.

In some embodiments, the cytoskeleton modulator and/or actin polymerization inhibitor and/or cytoskeleton polymerization modulator is a microfilament or actin stabilizer, polymer or polymerization inhibitor (e.g., cytochalasin family, alternative cytochalasin). ), latrunculin, jasplakinolide, phalloidin, swinholide, colchicine), intermediate filament stabilizer, polymer or polymerization inhibitor, microtubule stabilizer, polymer or polymerization inhibitors, lysophosphatidic acid, staurosporine, blebbistatin, Y27632, septin, and combinations thereof. These substances (cytoskeleton modulators and/or actin polymerization inhibitors and/or cytoskeleton polymerization modulators) are compounds that act directly or indirectly on the cytoskeleton (e.g., act upstream in a signaling pathway to affect myosin function). Y27632). As a non-limiting example, the addition of cytochalasin D can improve the mechanical properties and matrix deposition of stock purified, neocartilage engineered with multiple passaged chondrocytes. The present invention is not limited to the above-mentioned compounds.

These methods further include treating the cells with a cytoskeleton modulator, an actin polymerization inhibitor (e.g. cytochalasin D), a cytoskeleton polymerization modulator, or a combination thereof prior to treating the cells with the stock solution. can do.

In some embodiments, the cytoskeleton modulator, actin polymerization inhibitor, or cytoskeleton polymerization modulator acts directly or indirectly upstream in a signal transduction pathway. Cytoskeletal modulators inhibit, stabilize, or strengthen the cytoskeleton.

In some embodiments, cytochalasin D (or a cytoskeleton modulator, an actin polymerization inhibitor, and/or a cytoskeleton polymerization modulator) is applied during 0 to 48 hours during which new cartilage is formed.

In some embodiments, the stock solution is introduced after separation of cells from tissue, after thawing, after monolayer culture, after redifferentiation, or before formation of new tissue. The stock solution can be applied to the tissue using mechanical means or perfusion.

A method of treating a subject may include using the isolated retained cells directly for treatment.

This method may include further processing of the isolated retained cells by subculturing them as a monolayer in two dimensions.

These methods may include further processing the isolated retained cells into three-dimensional culture containing one or more of the following: 1) a suspension culture process, 2) a hydrogel, collagen gel, alginate, decellularized membrane, or Cellular tissue, dehydrated membrane or tissue, lyophilized membrane or tissue, hydroxyapatite of any stoichiometry, ceramics such as α-tricalcium phosphate, β-tricalcium phosphate, natural matrices such as silk, polylactic acid or polyester. Any shape or size, such as synthetic materials such as lactide (PLA), poly(lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG), polyglycolide (PGA), polycaprolactone, or combinations thereof. 3) support-free techniques such as self-assembly, pellet culture, aggregate culture, cell sheet, cell tissue fusion or a combination of any of these, 4) combination of support-free and support-based, 5) alone or other Process with type and treatment cells.

These methods may further include seeding the isolated retained cells (e.g., after pelleting). Cells can be seeded into non-adherent wells. This method may further include seeding cells (e.g., chondrocytes), such as pelleted cells, into non-adherent wells, wherein the cells seeded into the non-adherent wells form new cartilage. The present invention is not limited to the process of seeding cells into non-adherent wells.

In some embodiments, the resulting new cartilage has mechanical properties (e.g., aggregate modulus, shear modulus, tensile modulus, compressive stiffness, tensile stiffness, and tension) compared to new cartilage made from chondrocytes that have not been treated with a stock solution (e.g., ACK buffer). one or more of the intensity) increases. In another embodiment, the resulting new cartilage has a precise shape (e.g., flat, unrolled, etc.) compared to new cartilage made from chondrocytes that have not been treated with a storage solution (e.g., ACK buffer).

In some embodiments, the new cartilage improves new cartilage matrix synthesis and deposition compared to new cartilage made from chondrocytes that have not been treated with a depot. In some embodiments, new cartilage may have improved collagen cross-linking compared to new cartilage made from chondrocytes that have not been treated with a depot.

In some embodiments, the donor is a fetal donor, adolescent donor, or adult donor.

These methods may include further treatment of the isolated retained cells with chemical agents or bioactive substances. Non-limiting examples of these factors and substances include active and latent forms of growth factors (e.g., TGF superfamily, growth differentiation factors, osteogenic proteins), cytoskeletal regulators (cytochalasin D), and physiological activities. Substances, hormones (e.g. triiodothyronine, parathyroid hormone), mitogens, enzymes (e.g. chondroitinase-ABC, lysyl oxidase, Lysyl oxidase, lysyl oxidase-type 2), collagen cross-linking substances, toxic compounds, molecules that act upstream in the signal transduction pathway, or a combination of these.

These methods include molecules comprising one or more of SZP/PRG4, chondroitin sulfate, link protein, hyaluronic acid, keratin sulfate, dexmethane sulfate, and aggrecan, types I, II, III, V, VI, It may involve further treatment of the isolated retained cells with any substance that increases the production of the molecule.

These methods may involve further treatment of the isolated retaining cells with varying oxygen tensions achieved by environmental hypoxia or enzymatic conditions.

These methods may further include subjecting the cells to physical stimulation, such as static or dynamic direct compression, hydrostatic pressure, shear, tension, fluid flow induced shear, perfusion, or combinations thereof.

These methods may further include treating the separated retained cells with hyaluronidase in combination with a cytoskeleton modulating agent, an actin polymerization inhibitor, or a cytoskeleton polymerization modulator.

The method of the present invention enriches cell populations. This method can improve cell homogeneity. These methods can improve the strength of cell populations.

These methods may further include using the isolated retained cells in combination with other prepared cells and tissues.

This method can be applied to cells or cell tissues for the purpose of tissue engineering, for example, cartilage tissue engineering. This method can be applied to cells or tissue for the purpose of cell transplantation, for example, autologous chondrocyte transplantation (ACI). This method can be applied to tissue for the purpose of tissue transplantation, for example in mosaic plastic surgery.

It is not intended to limit the present invention to a specific theory or mechanism, and it is believed that the method and system of the present invention can improve the mechanical properties of neonatal tissue made from fetal-age cells to those of tissue made from adult-level cells. . It is not intended to limit the present invention to a specific theory or mechanism, and the method and system of the present invention appear to be advantageous because there is currently no standardized chondrocyte purification method.

The present invention is not limited to cells used in engineering applications. For example, the methods and systems of the present invention can be used in a variety of applications such as cancer cell applications, cell purification processes, and transplantation (e.g., fat transplantation). In some embodiments, methods for enriching cell populations of the invention provide preferred cell populations for use prior to or in preparation for treating a subject. Enhanced cells can be administered directly to the subject (post-enrichment use). Enriched cells can be further cultured in two dimensions prior to administration to subjects, including passage in monolayers (use after enrichment). Enriched cells can be further cultured in three dimensions, including suspension culture, prior to administration to subjects (post-enrichment use). Enriched cells can be further cultured in vitro for tissue engineering (post-enrichment use) using scaffold-free systems, including self-assembly, or scaffold-based systems, including natural and synthetic materials, before administration to subjects. Enhanced cells can be used for cell transplantation, transplantation and/or transplantation of tissue to treat a subject (use after enhancement). One or more of these post-strengthening uses may be performed after the strengthening method.

The present invention is not limited to cells used in engineering applications. For example, the methods and systems of the present invention can be used in a variety of applications such as cancer cell applications, cell purification processes, and transplantation (e.g., fat transplantation).

The stock solution can be introduced at any point in the culture, such as after monolayer culture, after redifferentiation, or before formation of de novo tissue, to create a cell population enriched with cells devoid of pre-existing undesirable cytoskeletal, membrane surface area, and rigidity properties. . As previously discussed, the present invention is not limited to ACK buffer.

As previously discussed, cytoskeleton modulators and/or actin polymerization inhibitors (e.g. cytochalasin D) and/or cytoskeleton polymerization modulators may be optionally applied. Example 4 below describes the application of cytochalasin D. For example, in some embodiments, 2 μM cytochalasin D may be applied for 0 to 48 hours during which new cartilage is formed through a self-assembly process. At this time, please note that the present invention is not limited to Example 4. Cytochalasin D can be used with other cartilage tissue engineering systems, such as autologous or scaffold-based systems, as well as chondrocytes from other sources, such as nasal or otic chondrocytes or osteoarthritis chondrocytes.

The methods described herein can be used independently or in combination. Purification treatments (e.g. stock solutions) and/or application of cytoskeletal modulating substances may be applied at different time points throughout the culture.

The present invention also provides tissue engineering, cell transplantation, or fat transplantation of various cellular tissues such as articular cartilage using purified cells. In some embodiments, the pelleted cells are for cell transplantation or tissue engineering, or transplantation. In some embodiments, pelleted cells are for cell injection.

In some embodiments, the methods of the present invention include: isolating cells from a donor, treating cells with a stock solution, pelleting cells, passage/proliferating cells in a monolayer, redifferentiating cells, and a process of seeding redifferentiated cells. Cells can be seeded into non-adherent wells (e.g., non-adherent agarose wells). The present invention is not limited to the process of seeding cells into non-adherent wells. Techniques for tissue engineering may be scaffold-based or scaffold-free.

In some embodiments, the methods of the present invention involve preparing neoplastic tissue made from fetal-age chondrocytes that have mechanical properties similar to adult articular cartilage.

These methods target cell populations with existing properties favorable for the formation of functional cells and/or de novo tissue, i.e., cells with an intact cytoskeleton capable of reorganization, cells with high membrane surface area, and unmodified rigidity (capable of structural changes). It may be intended to enrich a cell population, including but not limited to cells with This method may be aimed at improving cell populations to engineer native-like neocartilage. These methods may be aimed at improving cell populations to engineer new tissue similar to native ones.

In some embodiments, the methods of the present invention allow for the use of lower seeding densities (e.g., for the production of de novo tissue). For example, the method of the present invention improves the strength of the cell population so that fewer cells are required (e.g. compared to other methods). In some embodiments, a seeding density of approximately 2 million cells per construct is used. In some embodiments, using a seeding density of about 2 million cells per structure further increases the aggregate and shear moduli.

In the present invention, (i) a storage solution, (ii) a cytoskeleton regulator, an actin polymerization inhibitor (e.g. cytochalasin D), a cytoskeleton polymerization regulator, or a combination thereof, or (iii) a reservoir and cells. Note that additional biochemical treatments and/or mechanical stimulation may be used in combination with skeletal modulators, actin polymerization inhibitors (e.g. cytochalasin D), cytoskeletal polymerization modulators, or combinations thereof. For example, the present invention may provide: (A) Use of a stock solution to prepare cells for cell transplantation and/or tissue engineering in scaffold-free or scaffold-based systems: (i) the preparation may be physical; may involve the use of a stimulus (e.g. shear), (ii) the preparation may be further processed by biochemical treatment, (iii) the preparation may be subjected to further stimulation by mechanical means, (iv) the preparation may be subjected to further stimulation by biochemical treatment Additional processing and stimulation can be done by mechanical and mechanical means. (B) Use of cytochalasin D to strengthen engineered neocartilage (both scaffold-free and scaffold-based systems): (i) the preparation can be further processed by biochemical treatment; (ii) the preparation can be subjected to further stimulation by mechanical means, and (iii) preparations can be subjected to further processing and stimulation by biochemical and mechanical means. and (C) the combined use of stock solutions with cytochalasin D: (i) the preparation can be further processed by biochemical treatment, (ii) the preparation can be subjected to further stimulation by mechanical means, ( iii) The preparations can be subjected to further processing and stimulation by biochemical and mechanical means.

In summary, non-limiting examples of the present invention include (1) storage solution, (2) cytochalasin D, (3) storage solution + cytochalasin D, (4) storage solution + Biochemical treatment, (5) storage solution + physical stimulation, (6) storage solution + biochemical treatment + physical stimulation, (7) cytochalasin D + biochemical treatment, (8) cytochalasin D ( cytochalasin D) + physical stimulation, (9) cytochalasin D + biochemical treatment + physical stimulation, (10) storage solution + cytochalasin D + biochemical treatment, (11) storage solution + cytochalasin D + physical stimulation, (12) storage solution + cytochalasin D + biochemical treatment + physical stimulation. The cytochalasin D mentioned above can be replaced by a cytoskeleton regulator, an actin polymerization inhibitor, a cytoskeleton polymerization regulator, or a combination thereof.

The methods and systems of the invention (e.g., the use of a storage solution, the use of a cytoskeleton modulating agent and/or an actin polymerization inhibitor and/or a cytoskeletal polymerization modulating agent) can be used independently or in conjunction with each other or with other bioactive substances (e.g. For example, it can be used in conjunction with growth factors, chondroitinase ABC, lysyl oxidase-like 2) and physical/mechanical stimulation (e.g., direct compression, shear, hydrostatic pressure, tension). For example, it can be used to achieve greater functional properties of engineered de novo tissue (e.g., articular cartilage).

It is not intended to limit the present invention to a particular theory or mechanism, but it is advantageous to treat with a cytoskeleton regulator and/or an actin polymerization inhibitor (e.g. cytochalasin D) and/or a cytoskeleton polymerization regulator. This appears to be because it helps to induce compressive properties similar to native ones in engineered new cartilage. Specifically, multiple passaged fetal chondrocytes treated with cytochalasin D underwent self-assembly to form new cartilage with compression properties equivalent to native adult cartilage. This level of mechanical strength has never been previously observed in a fetal chondrocyte source.

The examples below describe the process of applying ACK buffer to fetal sheep and juvenile bovine chondrocyte isolates. This treatment resulted in significant improvements in the homogeneity, matrix deposition, and mechanical properties of the new cartilage construct.

For example, and not intended to limit the present invention to a particular theory or mechanism, during biopsy to obtain chondrocyte samples, cells from surrounding tissue or hematopoietic cells (e.g., apoptosis-inducing cells) may contaminate the sample. It appears that there is. Exposure of chondrocytes to hematopoietic cells (e.g., apoptosis-inducing cells) during or after cartilage sample collection can cause chondrocytes to enter an apoptotic or apoptotic state.

It is not intended to limit the present invention to any particular theory or mechanism, but rather that the purification process reduces contaminating cells (e.g., early apoptotic cells or apoptotic cells), particularly those with a weakened cytoskeleton, low membrane surface area, and high stiffness. It appears to be effective in increasing the functionality of therapeutic cells by reducing cell populations with existing undesirable properties, including but not limited to damaged cells.

Certain embodiments of the present specification, for example, the methods of the present specification, may include the process of collecting chondrocyte samples. In some embodiments, the chondrocyte sample includes a mixed population of apoptotic chondrocytes and non-apoptotic chondrocytes. In some embodiments, such methods include treating cells with a stock solution and selectively removing chondrocytes in early apoptotic stages. In some embodiments, the chondrocyte sample is more homogeneous after treatment with the stock solution.

Certain embodiments of the present disclosure, such as methods herein, may include collecting a cell sample derived from a portion of a rib. In some embodiments, the cell sample includes a mixed population of non-apoptotic cells and early apoptotic cells. In some embodiments, such methods include treating cells with a stock solution and selectively removing cells in early stages of apoptosis. In some embodiments, the cell sample is more homogeneous after treatment with the stock solution.

Certain embodiments herein, such as methods herein, may include collecting a human cell sample derived from a portion of a rib. In some embodiments, the human cell sample includes a mixed population of non-apoptotic human cells and early apoptotic human cells. In some embodiments, such methods include treating cells with a stock solution and selectively removing human cells in early apoptotic stages. In some embodiments, human cell samples are more homogeneous after treatment with the stock solution.

The present invention may provide a method of preparing a cell sample, the method comprising: (a) collecting a chondrocyte sample, wherein the chondrocyte sample is comprised of non-apoptotic early chondrocytes and early apoptotic chondrocytes; It includes a mixed population, and (b) includes the process of treating the chondrocyte sample of (a) with a storage solution, wherein the storage solution reduces early apoptotic cells in the sample. In some embodiments, this method may be repeated multiple times alone or in combination with other treatments. In some embodiments, the stock solution is ammonium chloride-potassium lysis buffer (ACK buffer). (b) Later chondrocyte samples have a higher proportion of non-apoptotic early cells compared to (b) pre-chondrocyte samples.

The present invention may also provide a method of preparing a cell preparation, the method comprising collecting a chondrocyte sample and subjecting the chondrocyte sample to a stock solution. In some embodiments, the stock solution is ammonium chloride-potassium lysis buffer (ACK buffer). In some embodiments, the cell preparation contains a higher percentage of non-apoptotic cells compared to the originally collected chondrocyte sample.

The present invention may also provide a method of preparing a cell preparation, the method comprising collecting a chondrocyte sample and processing the fully differentiated chondrocyte sample into a stock solution. In some embodiments, the stock solution is ammonium chloride-potassium lysis buffer (ACK buffer). This method improves the ability of the sample to form viable tissue.

The present invention also provides a method of preparing human chondrocyte samples. This method may include collecting a human chondrocyte sample from a portion of the rib and treating the human chondrocyte sample with a storage solution (e.g., ammonium chloride-potassium lysis buffer (ACK buffer)). This method may further include dissecting a portion of the rib prior to processing the human chondrocyte sample. In some embodiments, the process of dissecting a portion of a rib may remove cells unsuitable for tissue formation (e.g., to remove undesirable cell types). This method may further include enzymatically disintegrating a portion of the rib prior to processing the human chondrocyte sample. In some embodiments, the chondrocyte sample is a primary cell sample.

The present invention also provides production by a method comprising the steps of (a) collecting a cartilage sample and disintegrating the cartilage sample to obtain a cell suspension, and (b) treating the cell suspension with a stock solution to obtain a processed cell sample. Samples of treated cells may be provided. In some embodiments, the treated cell sample is suitable for producing new cartilage. In some embodiments, the chondrocyte sample is a primary cell sample. This method may further include centrifuging the processed cell sample after (b) and resuspending the cell sample.

The invention is not limited to the methods or compositions described herein.

[Example]

The following shows non-limiting examples of the invention. Additionally, it should be understood that the examples are not intended to limit the invention in any way, and equivalents or alternatives are within the scope of the invention.

A. Purification based on cytoskeletal properties

Example 1 - Stock solution

Example 1 describes a method of using stock solutions to select cells based on cytoskeletal properties. Example 1 shows that treatment of freshly isolated, fully differentiated cells with stock ACK buffer improves their ability to form viable tissues. In the following study, clinically relevant articular chondrocytes (ACs) from fetal and juvenile cartilage were used as models. Fetal sheep articular chondrocytes (foACs) were treated with ACK buffer during isolation. It is not intended to limit the present invention to a specific theory or mechanism, but rather that the process of treating chondrocytes with a storage solution increases the purity of viable chondrocytes by reducing the number of cells with existing undesirable cytoskeletal characteristics. It appears to be effective. Therefore, this treatment generates a cell population enriched with viable chondrocytes without undesirable cytoskeletal properties, thereby increasing the functional properties of the resulting self-assembled neocartilage. The effect of ACK buffer treatment was also investigated in cells derived from animal models of different species and ages, specifically juvenile bovine articular chondrocytes (jbACs).

Cell Isolation: foACs were harvested from the patellofemoral joints of three fetuses (120-125 days gestation) female Dorper cross sheep. jbACs were harvested from the patellofemoral joint surfaces of three juvenile (2-14 days old) male Holstein and Jersey calves. The tissue processing for sheep and cattle was the same. Articular cartilage from the entire surface of both condyles and trochlear grooves was minced into approximately 1 mm3 pieces and cultured in Dulbecco's modified Eagle's medium (DMEM with 4.5 g/L glucose and GlutaMAX; Gibco) and 2% (v/v) penicillin/streptomycin. /Punsixon (PSF; BD Biosciences) was washed three times and centrifuged (500 G, 5 minutes). Cell tissue was incubated with 0.2% (w/v) type II collagenase (Worthington) in DMEM containing 3% (v/v) fetal bovine serum (FBS; Atlanta Biologicals) for 18 h at 37°C with gentle shaking. Digested. After digestion, the resulting cell solution was passed through a 70 μm cell strainer, centrifuged (500 G, 5 min), and resuspended in colorless DMEM. ACs and RBCs were counted, and the viability of ACs was assessed by trypan blue staining. Half of the foACs and jbACs were treated with ACK buffer as detailed below. After treatment with ACK buffer, cells were counted again and viability was assessed. Untreated cells were washed with colorless DMEM instead of ACK buffer, but otherwise treated identically. The cells immediately underwent self-assembly.

ACK buffer treatment: ACK buffer consisted of 154.4 mM ammonium chloride (Sigma), 10 mM potassium bicarbonate (Sigma-Aldrich), and 97.3 μM ethylenediaminetetraacetic acid (EDTA) tetrasodium salt (Acros Organics). This corresponds to 8.26 g ammonium chloride, 1.0 g potassium bicarbonate and 0.037 g EDTA in 1 L of ultrapure water. This solution was sterile filtered before use.

Protocol for introducing ACK buffer to purify chondrocytes: (1) ACK buffer was warmed to 37°C. (2) Up to 100 million chondrocytes were dispensed into a 50 mL conical tube. (3) The cell solution was centrifuged at 500 G for 5 minutes. (4) Aspirate the supernatant, gently resuspend the cell pellet in 10 mL of ACK buffer, and incubate at 37°C for 3 to 5 minutes. (5) The ACK buffer cell suspension was centrifuged at 500 G for 5 minutes. (6) ACK buffer was aspirated. Cell pellets were washed twice with colorless or washing medium before dish incubation or freezing.

New cartilage structure seeding and culture: Primary foACs and jbACs treated with ACK buffer (+ACK Treatment) and untreated (-ACK Treatment) foACs and jbACs were self-assembled into engineered new cartilage structures in non-adhesive agarose wells, respectively. . A sterile stainless steel mold consisting of a 5 mm diameter cylindrical post was inserted into a 48-well plate containing 1 mL of molten 2% (w/v) molecular biology grade agarose (Thermo) in each well to add a single agar to each plate well. Roswell was created. After the agarose solidified at room temperature, the mold was removed. Chemically defined chondrogenic medium (CHG medium) (1% PSF, 1% ITS+ premix (BD Biosciences), 1% non-essential amino acids (Gibco), 100 nM dexamethasone (Sigma), 50 mg/mL Aspergillus agarose was added to the agarose wells. DMEM containing cobic acid-2-phosphate (Sigma), 40 g/mL L-proline (Sigma), and 100 mg/mL sodium pyruvate (Sigma) was charged. CHG medium was changed twice during 5 days before cell seeding to ensure agarose saturation. 4.5 million cells of treated and untreated foACs and jbACs, respectively, were seeded in 100 μL CHG medium in 5 mm agarose wells. Constructs were separated on day 6 and placed into larger wells coated with agarose to prevent adhesion of constructs to the wells. During the 6-week culture period, the medium was changed every day before the restrictions were lifted and every 2 days after the restrictions were lifted. At the end of the culture period, gross morphological analysis, histology, immunohistochemistry (IHC), glycosaminoglycans (GAGs) and collagen quantification, and mechanical evaluation were performed.

Gross morphology analysis: The thickness of the structures was measured from photographs of the structures using ImageJ software (National Institutes of Health). Wet weights were obtained by weighing the entire construct before splitting the sample for histological, biochemical, and mechanical analysis.

Histological and immunohistochemical (IHC) evaluation: Samples were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at 5 μm thickness along the minor axis to expose the entire thickness of the construct. Sections were stained with H&E to show morphology, stained with Safranin O/Fast Green to visualize glycosaminoglycans (GAGs), and stained with Picrosirius red to visualize collagen. Additionally, IHC was performed on collagen I (ab90395, 1:250 dilution, Abcam) and collagen II (ab34712, 1:4000 dilution, Abcam).

Biochemical evaluation: For biochemical analysis, the wet weight of the divided structure sample was measured, freeze-dried, and weighed again to determine the dry weight. The weight difference before and after freeze-drying was quantified as the sample water weight to calculate the structure hydration. Lyophilized samples were digested with 125 μg/mL papain (Sigma-Aldrich) for 18 h at 65°C. Glycosaminoglycan (GAG) content was quantified using the Blyscan assay kit (Biocolor). Collagen content was quantified using a modified colorimetric chloramine-T hydroxyproline assay. A standard curve was generated using Sircol collagen standard (Biocolor). DNA content was quantified using PicoGreen dsDNA reagent (Invitrogen). Collagen and glycosaminoglycan (GAG) content was quantified by wet weight, dry weight, and DNA content.

Mechanical Evaluation: Creep indentation compression tests were performed on 3 mm diameter punches for each structure. A 0.8 mm diameter flat, porous indenter tip was applied to the sample using masses ranging from 0.45 to 2 g to achieve a strain of 10 to 15%. Collective elastic modulus and shear modulus were obtained from experimental data using semi-analytical and quasi-numerical linear ideal models and finite element models. For tensile testing, samples were punched into dog bone-shaped specimens with a gauge length of 1.92 mm according to ASTM standards (ASTM D3039). A paper tab was glued to the sample outside the gauge length, secured in a TestResources instrument (TestResources Inc.), and pulled at a rate of 1% of the gauge length per second until the sample was broken. The cross-sectional area of the sample was measured with ImageJ and used to generate a stress-strain curve. The tensile modulus was obtained by a least squares fit to the linear region of the curve. The maximum stress represented the ultimate tensile strength (UTS).

Statistical analysis: Biochemical and mechanical data were analyzed using the Student t-test in Prism 6 (GraphPad Software). A p-value of less than 0.05 indicates statistical significance, and the sample size per group was n = 6. In figures displaying quantitative results, groups that are not indicated with the same symbol indicate statistical differences, and all data are expressed as mean ± standard deviation.

Results: Figure 1 shows the morphology of the separated cell pellet and number of cells before and after ACK buffer treatment. ACK buffer treatment resulted in a change in the morphology of the pellet in both cell types. Before processing, the foAC pellets were bright red overall, and after processing, they were milky white. Before treatment, the jbAC pellets were pinkish-tan, and after treatment, they were milky white. The viability of foACs before and after treatment was 84 ± 11% and 82 ± 7%, respectively. The viability of jbACs before treatment was 92 ± 7% and after treatment it was 86 ± 3%. ACK treatment reduced the total cell number of foACs and jbACs by 19 ± 7% and 9 ± 3%, respectively. RBC content significantly decreased after treatment in both foACs (36 ± 14% before treatment, 14 ± 3% after treatment) and jbACs (21 ± 6% before treatment, 7 ± 2% after treatment).

Figure 2 shows the overall shape of the self-assembled new cartilage structure after culture for 6 weeks. All structures appeared hyaline-like with similar diameters. Spherical, diffuse areas (representing areas of “bad” cells that are unable to produce functional cartilage; these “bad” cells may have exhibited a fragmented/inactive cytoskeleton, reduced membrane surface area, and/or altered cell stiffness) This was present in both foAC and jbAC untreated groups. ACK treatment removed these areas and generated flat foAC and jbAC new cartilage. ACK treatment also reduced both the thickness and functional weight of foAC and jbAC new cartilage constructs. The thickness of foAC new cartilage was 1.2 ± 0.1 mm without treatment and significantly decreased to 0.7 ± 0.1 mm with treatment. The thickness of jbAC new cartilage was 0.58 ± 0.1 mm without treatment and significantly decreased to 0.38 ± 0.1 mm with treatment. The wet weight of foAC new cartilage was 26.6 ± 0.8 mg when untreated and significantly decreased to 15.1 ± 0.6 mg when treated. The wet weight of jbAC new cartilage was 13.3 ± 0.4 mg when untreated and significantly decreased to 7.3 ± 0.2 mg when treated. Hydration of foAC new cartilage was 87.1 ± 0.5% without ACK treatment and 87.2 ± 0.4% with ACK treatment. Hydration of jbAC new cartilage was 89.0 ± 0.3% without ACK treatment and significantly decreased to 86.4 ± 0.9% with ACK treatment.

Figure 3 shows histology and immunohistochemical staining of new cartilage structures after culture for 6 weeks. Histological examination revealed the presence of diffuse areas of low cell density and rich in glycosaminoglycans (GAG) in both untreated foAC and jbAC new cartilage. ACK treatment removed these diffuse areas, producing a homogeneous tissue that stained more intensely for glycosaminoglycan (GAG) and collagen in both foAC and jbAC constructs. There was strong glycosaminoglycan (GAG) staining in all groups, which was further increased by ACK treatment in both foAC and jbAC constructs. Collagen staining was present in all groups but was further enhanced by ACK treatment in both foAC and jbAC constructs. Collagen I staining was absent in both untreated and treated foAC and jbAC constructs. Collagen II staining was present in both untreated foAC and jbAC constructs and was enhanced by ACK treatment.

Figure 4 shows the biochemical content of the new cartilage structure. The glycosaminoglycan (GAG) per water weight (glycosaminoglycan (GAG)/water weight) of untreated and ACK-treated foAC new cartilage was 5.5 ± 0.1% and 5.7 ± 0.2%, respectively. Glycosaminoglycan (GAG) per dry weight (glycosaminoglycan (GAG)/dry weight (DW)) of untreated and ACK-treated foAC new cartilage was 42.8 ± 1.5% and 43.1 ± 1.7%, respectively. Glycosaminoglycan (GAG) per DNA of the untreated foAC construct was 60.4 ± 0.9 μg/μg, and significantly decreased to 50.54 ± 1.3 μg/μg upon ACK treatment. ACK treatment decreased the glycosaminoglycan (GAG) per wet weight of the jbAC construct from 3.9 ± 0.2% to 3.0 ± 0.1%, and the glycosaminoglycan (GAG) per dry weight from 33.5 ± 2.0% to 24.8 ± 2.8%. significantly decreased. ACK treatment significantly reduced glycosaminoglycan (GAG) per DNA of the jbAC construct from 70.65 ± 5.3 μg/μg to 28.1 ± 1.4 μg/μg.

The collagen content per wet weight (collagen/wet weight) and collagen content per dry weight (collagen/dry weight (DW)) of foAC new cartilage increased from 2.0 ± 0.1% to 2.3 ± 0.1% and 14.4 ± 0.8%, respectively, by ACK treatment. increased significantly to 18.5 ± 0.7%. Collagen per structural DNA of untreated and ACK-treated foAC new cartilage was 20.5 ± 0.9 μg/μg and 20.4 ± 0.8 μg/μg, respectively. ACK treatment significantly increased collagen per wet weight of the jbAC construct from 1.8 ± 0.1% to 2.0 ± 0.1%. Collagen per dry weight of the untreated jbAC construct was 15.2 ± 0.5% and that of the ACK-treated construct was 16.3 ± 1.4%. Collagen per DNA of the untreated jbAC construct was 31.7 ± 1.2 μg/μg, and significantly decreased to 18.6 ± 0.7 μg/μg upon ACK treatment.

Figure 5 shows the mechanical properties of the new cartilage structure. ACK treatment significantly improved the compressive, shear, and tensile properties of both foAC and jbAC neocartilage constructs. The collective elastic modulus of the foAC structure significantly increased from 37.8 ± 8.1 kPa to 104.5 ± 13.5 kPa by ACK treatment. ACK treatment similarly significantly increased the collective modulus of the jbAC structure from 83.8 ± 7.0 kPa to 116.6 ± 8.8 kPa. The shear modulus of foAC and jbAC new cartilage significantly increased from 21.6 ± 3.5 kPa to 49.4 ± 6.4 kPa and from 38.5 ± 3.3 kPa to 51.9 ± 4.0 kPa, respectively, by ACK treatment. ACK treatment significantly increased the tensile modulus of the foAC structure from 0.8 ± 0.1 MPa to 1.5 ± 0.1 MPa and the ultimate tensile strength (UTS) from 0.2 ± 0.1 MPa to 0.5 ± 0.1 MPa. The tensile modulus of the jbAC structure significantly increased from 1.2 ± 0.1 MPa to 1.8 ± 0.1 MPa, and the UTS significantly increased from 0.6 ± 0.1 MPa to 1.1 ± 0.1 MPa as a result of ACK treatment.

Example 2 - Shearing

Example 2 describes a method of using shear to select cells based on cytoskeletal properties. Example 2 shows a protocol for purifying articular chondrocytes by applying shear.

Cell Isolation: Juvenile sheep joint chondrocytes (joACs) are isolated from the femoral condyles and trochlear grooves of juvenile Rambouillet Suffolk crossbred sheep obtained from a local abattoir (Nature's Bounty Farms, Dixon, CA) within the day of animal sacrifice. Cartilage is chopped into 1-2 mm ³ pieces and washed twice with washing medium (Dubelco's Modified Eagle Medium; DMEM containing 1% (v/v) PSF). Minced cartilage was incubated with 500 units/mL collagenase II in chondrogenesis medium + 3% (v/v) fetal bovine serum (FBS; Atlanta Biologicals) for 18 h at 37°C and 10% CO2 on an orbital shaker. Digest with a mold (Worthington Biochemical). The cells are then filtered through a 70 μm filter and counted.

Protocol to introduce shear to purify chondrocytes: (1) Place approximately 30 mL of cell solution into a conical tube. (2) Attach a cone filter containing a 15-20 μm sized mesh so that the vacuum forces the flow of cell solution into the new cone tube. (3) Attach a new conical tube to the other side of the vacuum filter and attach the filter to the vacuum line. (4) Invert the cone tube and filter set so that the cell solution flows through the filter and into the new cone tube. Wait until all the solution has passed through the filter. (5) Separate the filter and the old conical tube. The filtered cell solution is washed twice with washing medium and the remaining cells are counted.

Example 3 - Impact/Compression

Example 3 describes a method of using shock/compression to select cells based on cytoskeletal properties. Example 3 shows a protocol for purifying articular chondrocytes by applying compression/impact.

Cell Isolation: Juvenile sheep articular chondrocytes (joACs) obtained from a local abattoir (Superior Farms, Dixon, CA) within 48 hours after slaughter were isolated from the patellofemoral surface of one-year-old Rambouillet Suffolk crossbred sheep (n = 8). The cartilage of the surfaces of both condyles and trochlear grooves was minced into approximately 1 mm ³ pieces and cultured in Dulbecco's modified Eagle's medium (DMEM containing 4.5 g/L glucose and GlutaMAX; Gibco) and 2% (v/v) penicillin/streptomycin/fern. Wash three times with conventional (PSF; Lonza). The cartilage was then incubated with 0.2% (w/v) type II collagenase (Worthington) in DMEM containing 3% (v/v) fetal bovine serum (FBS; Atlanta Biologicals) for 18 h at 37°C with gentle shaking. digests. After digestion, the resulting cell solution is filtered through a 70 μm cell strainer.

Protocol to introduce compression/impact to purify chondrocytes: (1) Place approximately 30 mL of cell solution into the conical tube. (2) Place 5 glass beads with a diameter of 0.5 to 1.25 mm into a test tube. (3) Gently roll the cone tube on the plate rocker for 3 minutes. (4) Pipette the cell solution into a new cone tube. Wash the glass beads three times with washing medium and add this washing solution to a new conical tube. (5) Wash the treated cell solution twice with washing medium and count the remaining cells.

B. Purification based on membrane surface area properties

Example 4 - Stock solution

Example 4 describes a method of using stock solutions to select cells based on membrane surface area properties. Example 4 shows that native-like new cartilage can be achieved using multiple passaged chondrocytes. The invention is not limited to the methods or compositions described herein. In Example 4, a cartilage engineering model of the self-assembly process was used. It is not intended to limit the present invention to a specific theory or mechanism, but rather that the process of treating primary chondrocytes with a storage solution increases the purity of viable chondrocytes by reducing the cell population with existing undesirable membrane surface area characteristics. It appears to be effective. Therefore, it appears that this treatment increases the functional properties of the resulting self-assembled neocartilage by generating a cell population enriched with viable chondrocytes free of pre-existing undesirable membrane surface properties.

Example 4 mimics cell proliferation (chondrogenically coordinated expansion), condensation, differentiation (aggregate redifferentiation culture), cartilaginous matrix production (self-assembly), and matrix maturation in vitro (purified into stock solution and then extensively (using passaged chondrocytes) produces new cartilage with mechanical properties equivalent to native articular cartilage from which the cells were derived. Example 4 describes three steps. In step 1, the seeding density was determined for both primary and passaged/redifferentiated chondrocytes, i.e., the seeding density that would produce neocartilage constructs with maximum functional properties (and the culture system that required the minimum number of chondrocytes was selected). ). It is not intended to limit the present invention to any particular theory or mechanism, but rather that mimicking the developmental sequence of chondrogenically coordinated cell expansion, aggregation and aggregate redifferentiation under optimized culture conditions can lead to neogenesis from primary cells from purified multi-passage cells. It appears that new cartilage equivalent to cartilage is produced. In step 2, the usefulness of cytochalasin D and hyaluronidase treatment to further promote chondrogenic redifferentiation of expanded chondrocytes was determined. Without intending to limit the present invention to a particular theory or mechanism, it appears that the combined treatment promotes the production of cartilage-specific matrix and increases the functional properties of the new cartilage construct. In the third stage, matrix formation and cross-linking-based maturation were promoted in new cartilage. It is not intended to limit the present invention to a specific theory or mechanism, and TGF- 1, Treatment with c-ABC and LOXL2 appears to improve the functional properties of new cartilage equivalent to that of cell-derived native articular cartilage.

Chondrocyte isolation: Fetal sheep articular chondrocytes (foACs) were harvested from the patellofemoral surface of Dorper crossbred sheep at 120 days of gestation obtained from medical waste (UC Davis School of Veterinary Medicine). The entire surface of both condyles and the cartilage of the trochlear groove were cut into approximately 1 mm ³ pieces and then cultured in Dulbecco's modified Eagle's medium (DMEM with 4.5 g/L glucose and GlutaMAX; Gibco) and 2% (v/v) penicillin/streptomycin. /Washed 3 times (500 G, 5 minutes) with Punji Zone (PSF; Lonza) and centrifuged. Cell tissue was incubated with 0.2% (w/v) type II collagenase (Worthington) in DMEM containing 3% (v/v) fetal bovine serum (FBS; Atlanta Biologicals) for 18 h at 37°C with gentle shaking. Digested. The cell solution produced after digestion was filtered through a 70 μm cell strainer. For studies 1-3, foACs were incubated in ACK buffer (3 min in ultrapure water) containing 154.4mM ammonium chloride (Sigma), 10mM potassium bicarbonate (Fisher Scientific), and 50mM EDTA tetrasodium salt (Acros Organics), as previously described. Washed with . These primary (P0) foACs were frozen in DMEM containing 20% (v/v) DMSO (Sigma) and 10% (v/v) FBS.

Chondrocyte expansion and redifferentiation: Previously frozen P0 foACs were seeded into T-225 flasks at a density of ^1.5x104 cells/ ^cm2 and supplemented with 2% FBS and chondrogenically adjusted TFP (1 ng/mL TGF- 1, 5 ng/mL bFGF, 10 ng/mL PDGF; Chemically defined chondrogenic medium (CHG medium) containing (1% PSF, 1% ITS+ premix (BD Biosciences), 1% essential amino acids (Gibco), 100 nM dexamethasone (Sigma), 50 mg/mL, all from PeproTech). (DMEM containing mL ascorbate-2-phosphate (Sigma), 40 g/mL L-proline (Sigma), and 100 mg/mL sodium pyruvate (Sigma)). The medium was changed every 23 days. At fusion, cells were lifted with 0.5% Trypsin-EDTA (Gibco) for 5 minutes and then incubated with DMEM containing 0.2% type II collagenase and 2% FBS at 37°C for approximately 1 hour with stirring every 20 minutes. The cell layer was digested. The resulting cell solution was filtered through a 70 μm cell strainer and reseeded in T-225 flasks to achieve passage 3 (P3). P3 foACs underwent aggregate redifferentiation (P3R) as previously described. In summary, TGB supplementation (10 ng/mL TGF- 1, 100 ng/mL GDF-5, 100 ng/mL BMP-2; CHG medium containing 750,000 cells/mL (all from PeproTech) was plated on 100 mm x 20 mm Petri coated with 1% (w/v) molecular biology grade agarose (Thermo Fisher Scientific) made in phosphate-buffered saline (PBS; Sigma). Cultured in a dish to create a non-adherent environment. Aggregate cultures were maintained on an orbital shaker at 60 rpm for the first 3 days and static for the remaining 14 days of redifferentiation. The medium was changed every 23 days. At the end of the incubation period, the aggregates were digested with 0.5% Trypsin-EDTA for 20 minutes and then stirred every 20 minutes for approximately 2 hours at 37°C with 0.2% collagenase in DMEM containing 2% FBS. After the aggregates were dissociated, cells were filtered through a 70 μm cell strainer and counted.

Chondrocyte actin visualization: untreated P0 foACs and cytochalasin D treated and untreated P3 and P3R foACs to visualize the effect of cytochalasin D treatment on cytoskeleton-mediated chondrogenic redifferentiation in stage 2. F-actin staining was performed. Approximately 8x10 ³ cells/cm ² were attached to a glass slide for 1 hour in the presence of 2% FBS. Non-adherent cells were removed by exchanging PBS twice, and adhered cells were fixed with 3.9% formaldehyde in PBS for 10 minutes. After washing twice again with PBS, the fixed cells were permeabilized with 0.1% Triton-X 100 (Sigma) in PBS for 5 minutes. After washing twice with PBS, cells were stained with CF594-conjugated phalloidin (Biotium; diluted 1:200 in PBS) for 30 minutes. Excessive staining was removed by two exchanges with PBS, and cells were counterstained with Vectashield (Vector Laboratories) containing DAPI, covered with cover glasses, and visualized using the Texas Red fluorescence channel.

New cartilage construct seeding and culture: P0, P3, or P3R foACs were self-assembled into engineered new cartilage constructs in non-adherent agarose wells. A sterile stainless steel mold consisting of a 5 mm diameter cylindrical post was inserted into a 48 well plate containing 1 mL of molten 2% (w/v) agarose in each well, creating a single agarose well per plate well. After the agarose solidified at room temperature, the mold was removed. Agarose wells were filled with CHG medium, which was exchanged twice for 5 days before seeding, to ensure agarose saturation. At each step, cells were seeded in 5 mm agarose wells in 100 μL CHG medium per well. In step 1, P0 or P3R foACs were seeded at five densities (2, 3, 4, 5, and 6 million cells/construct), respectively. In step 2, P3 and P3R foACs were seeded at 2 million cells per construct. In the third stage, 2 million P3R foACs were sown. All constructs were isolated on day 6 and placed into larger wells coated with agarose to prevent construct attachment. During the 6-week culture period, the medium was changed every day before separation and every 2 days after separation. In stage 1, no chemical treatment was applied during neocartilage culture. In step 2, cytochalasin D (Enzo Life Sciences; 2 μM at sowing and for the first 48 hours) and hyaluronidase (200 units/mL at sowing) were applied in a full-full factorial design. did. In step 3, cytochalasin D was applied as in the previous step, and TGF- 1 (10 ng/mL for the entire culture period), chondroitinase ABC (c-ABC, Sigma; 2 units/mL for 4 h on day 7), and LOX cocktail (applied on days 7 to 21; lysyl oxidase-like TCL treatment consisting of 2 (LOXL2, Signal Chem; 0.15 μg/mL), copper sulfate (Sigma; 1.6 μg/mL), and hydroxylysine (Sigma; 0.146 μg/mL) was applied. For reference, P0 foACs (P0 control) that were not treated with ACK buffer during isolation were also seeded at 4.5 million cells per construct and were not subjected to any other chemical treatment during the new cartilage culture. All new cartilage assessments were performed at the end of the culture period.

Analysis of the overall shape of new cartilage: The diameter and thickness of the new cartilage structure were measured from photographs using ImageJ (National Institutes of Health). The wet weight was obtained by weighing the entire construct before splitting the samples for histological, biochemical, and mechanical analysis.

Histological and immunohistochemical evaluation of new cartilage: Formalin-fixed samples were embedded in paraffin and sectioned into 5 μm sections along the minor axis to expose the entire thickness of the construct. In all studies, morphology was demonstrated by staining with H&E, glycosaminoglycan (GAG) deposition was demonstrated with safranin O/fast green, and collagen was visualized with picrosirus red. In addition, Von Kossa and alizarin red staining were performed to confirm mineralization. Immunohistochemistry (IHC) was performed to stain collagen I (Abcam ab34710, dilution 1:250) and collagen II (Abcam ab34712, dilution 1:4000). In step 1, IHC was also performed to stain collagen VI (Abcam ab6588, dilution 1:250) and collagen X (Abcam ab49945, dilution 1:200).

Biochemical evaluation of new cartilage: The wet weight of the biochemical sample was measured, freeze-dried, and weighed again to determine the dry weight. Dried samples were digested in 125 μg/mL papain (Sigma-Aldrich), 5 mM N-acetyl-L-cysteine, 5 mM EDTA, 100 mM phosphate buffer for 18 h at 65°C. Glycosaminoglycan (GAG) content was measured using a Blyscan assay kit (Biocolor). Collagen content was measured by a modified colorimetric chloramine-T hydroxyproline assay using hydrochloric acid. A standard curve was generated using Sircol collagen standard (Bicolor). DNA content was measured using PicoGreen dsDNA reagent (Invitrogen). Collagen and glycosaminoglycan (GAG) content of new cartilage was quantified in terms of wet weight, dry weight, and DNA content. Pyridinoline cross-linking was quantified by high-performance liquid chromatography (HPLC) using pyridinoline standards (Quidel) as previously described. Pyridinoline content was quantified based on water content and collagen content.

Mechanical evaluation of new cartilage: Creep indentation compression tests were performed on each construct on a 3 mm diameter punch, flat and porous indenter using loads ranging from 0.45 to 2 g to achieve a strain of 10 to 15%. A tip (0.8 mm diameter) was applied. Collective and shear moduli were obtained from experimental data using semi-analytical and quasi-numerical linear ideal models and finite element analysis. Tensile testing was performed according to ASTM standards (ASTM D3039). The structure was punched out of a dog bone-shaped specimen with a gauge length of 1.92 mm, and paper tabs were glued to the tissue outside the gauge length. A paper tab was secured to a TestResources apparatus (TestResources Inc.) and pulled at 1% of the gauge length per second until the sample was broken. A stress-strain curve was generated from the experimental data, and the cross-sectional area of the sample was measured through ImageJ analysis. The tensile modulus was obtained using a least squares fit to the linear region of the curve, and the maximum stress represented the ultimate tensile strength (UTS).

Functional index evaluation: Engineered neocartilage was quantitatively evaluated against native fetal and juvenile bovine articular cartilage at all stages using a modified functional index (FI; Equation 1), and culture conditions to proceed to each stage were selected. All factors were given equal weight based on the structure-function relationship within cartilage, the importance of compressive and tensile properties during joint loading, the importance of biochemical properties for cellular tissue integrity, and the contribution of cross-linking to mechanical integrity. In the functional index, G is glycosaminoglycan (GAG)/water weight (%), C is total collagen/water weight (%), P is pyridinoline/collagen (nmol/mg), and E ^C is (compressed) The collective elastic modulus, E ^T represents the tensile elastic modulus. The subscripts nat and eng indicate native cell organization and designed cell organization, respectively. Structures with irregular thickness and abnormal shapes such as rupture, breakage, or spherical areas were considered unsuitable and excluded from the functional index evaluation.

Equation 1:

Statistical analysis: In step 1, quantitative new cartilage properties and functional indices were analyzed at different seeding densities across the two passage conditions using two-way analysis of variance (ANOVA) and Tukey's post hoc test in Prism 7 (GraphPad Software). In the second step, one-way ANOVA and Tukey's post hoc test were performed to analyze quantitative new cartilage properties and functional indices between different treatment groups. In the third step, Student t-test was performed to analyze quantitative characteristics and functional indices between treatment groups. A sample size of n = 6 per group was used. All data are expressed as mean ± standard deviation. Significance was determined at P < 0.05, and in figures displaying quantitative results, statistically different groups are indicated by different symbols.

Stage 1: The new cartilage structure showed morphological differences depending on passage and cell density (see Figure 6). In the case of P0 new cartilage, as the cell seeding density increased, the diameter, thickness, and water weight of the structure increased. The diameters of P3R constructs seeded at the same cell density as those of P0 constructs seeded with 2, 3, 4, 5, and 6 million cells were 5.3 ± 0.2 (Figure 6E), 6.2 ± 0.2 (Figure 6D), and 6.9 ± 6.9, respectively. 0.2 (Figure 6C), 7.1 ± 0.3 (Figure 6C), 7.2 ± 0.1 (Figure 6C), 8.2 ± 0.2 (Figure 6A), 8.2 ± 0.1 (Figure 6A, Figure 6B), 7.8 ± 0.3 (Figure 6B), 7.2 ± 0.1 (Figure 6C) and 7.0 ± 0.2 (Figure 6C) mm. The diameters of P3R constructs seeded at a cell density equal to the thickness of P0 constructs seeded with 2, 3, 4, 5, and 6 million cells were 0.5 ± 0.0 (Figure 6F), 0.5 ± 0.0 (Figure 6F), and 0.7 ± 0.7, respectively. 0.1 (Figure 6E), 0.7 ± 0.2 (Figure 6D, Figure 6E), 0.9 ± 0.1 (Figure 6B, Figure 6C), 0.9 ± 0.0 (Figure 6B, Figure 6C, Figure 6D), 1.0 ± 0.0 (Figure 6B), 1.2 ± 0.1 (Figure 6A), 0.7 ± 0.0 (Figure 6C, Figure 6D, Figure 6E), and 0.8 ± 0.1 (Figure 6B, Figure 6C, Figure 6D, Figure 6E) mm. The water weight of P0 constructs seeded with 2, 3, 4, 5, and 6 million cells and the diameter of P3R constructs seeded at the same cell density were 12.8 ± 0.5 (Figure 6G), 19.0 ± 0.6 (Figure 6F), and 30.2, respectively. ± 3.5 (Figure 6E), 35.2 ± 5.2 (Figure 6D), 39.5 ± 1.9 (Figure 6D), 49.5 ± 1.9 (Figure 6C), 53.6 ± 1.4 (Figure 6B, Figure 6C), 58.2 ± 1.3 (Figure 6A, Figure 6C). 6B), 59.3 ± 3.6 (Figure 6A), and 58.4 ± 4.0 (Figure 6A) mg. Constructs seeded with 2, 3, and 4 million cells were uniform, disk-shaped, and maintained a constant thickness within each construct. The structures with 2 and 3 million cells had a constant thickness but were curved, while the structures with 4 million cells were flat. Constructs seeded with 5 and 6 million cells showed inconsistent thickness and irregular morphology with folded and fractured edges. In P3R new cartilage, generally, as the seeding density increased, the diameter of the structure decreased and the thickness and wet weight increased. Constructs seeded with 4 million cells showed small, well-defined, diffuse matrix pockets of low cell density. At seeding densities of 5 and 6 million cells, this region ruptured and the structure formed two distinct layers, with only one layer remaining completely intact. The reported thickness of these structures was measured in intact layers.

Histologically, differences in cell morphology and glycosaminoglycan (GAG), collagen, and collagen II staining intensity were observed as a function of passaged and new cartilage seeding density (see Figure 6). H&E staining showed larger chondrocytes in both P0 and P3R constructs than cells present in native tissue. Additionally, the cavity surrounding the cells in the P3R structure was larger than that of P0 new cartilage. Safranin O staining for sulfated glycosaminoglycans (GAGs) showed stronger staining in both P0 and P3R constructs compared to native tissue. Safranin O stained less intensely in the outer region compared to the central region of P0 structures. This external region was greatly reduced in the P3R construct. Glycosaminoglycan (GAG) staining appeared strongest at a seeding density of 4 million cells in P0 new cartilage. Glycosaminoglycan (GAG) staining in P3R neocartilage was strongest at a density of 2 million cells and decreased with increasing seeding density. Picrosirius red staining for collagen was less intense in both P0 and P3R neoplastic tissue compared to native tissue. The outer regions of P0 and P3R structures stained more intensely than the inner regions, and these regions were thinner in P3R neocartilage. Picrosirius red staining was strongest at a seeding density of 4 million cells in P0 new cartilage and 2 million cells in P3R new cartilage. Collagen I staining was minimal in all groups. Collagen II staining within P0 new cartilage peaked at a seeding density of 4 million cells. Within P3R neocartilage, collagen II staining was strongest at a density of 2 million cells and decreased with increasing seeding density. Additional staining for collagen VI, collagen X, alizarin red and Von Kossa is shown in Figure 7. Both P0 and P3R new cartilage stained positive for collagen VI, and P3R new cartilage stained most darkly. P0 and P3R new cartilage also stained weakly for collagen New cartilage at all passages and seeding densities was not stained with alizarin red or Von Kossa. The biochemical content, mechanical properties and functional index calculations are shown in Table 1 and Figure 6 in Figure 13. The functional index confirmed that the optimal P0 (P0 Opt) and P3R (P3R Opt) seeding densities were 4 million and 2 million cells/construct, respectively. Based on the excellent functional index, the P3R group seeded at 2 million cells/construct was carried out in two stages.

For reference, new cartilage cultured in P0 foACs that was not treated with ACK buffer during isolation was also mechanically tested. Based on the methods of P0 foACs and previous studies, these constructs were seeded at a density of 4.5 million cells per construct. The collective modulus, shear modulus, and permeability were 97.7 ± 20.4 kPa, 43.1 ± 12.1 kPa, and 45.1 ± 15.7 x 10 ¹⁵ m ⁴ /Ns, respectively. The tensile modulus and UTS were 0.8 ± 0.2 MPa and 0.2 ± 0.1 MPa, respectively.

Phase 2: The first study in this phase examined cytochalasin D and hyaluronidase to determine the ability of chemical treatments to redifferentiate passaged chondrocytes without using aggregate redifferentiation. did. The P3 new cartilage structure showed significant morphological differences from the P3R new cartilage. In P3 new cartilage (see Figure 12), cytochalasin D (Cyto D) treatment resulted in a uniquely flat and uniform structure. Untreated, hyaluronidase treatment (Hya), or dual treatment (Hya + Cyto D) resulted in round new cartilage with diffuse pores in the center of the construct. The diameters of untreated, cytochalasin D-treated, and double-treated structures were 2.8 ± 0.2, 3.7 ± 0.2, 2.6 ± 0.1, and 3.4 ± 0.2 mm, respectively. The diameter of cytochalasin D-treated new cartilage was significantly larger than that of untreated, hyaluronidase-treated, and double-treated new cartilage. The diameter of the double-treated new cartilage was also significantly larger than that of the untreated and hyaluronidase-treated new cartilage. The thickness of new cartilage produced without treatment, hyaluronidase treatment, or dual treatment was 2.1 ± 0.2, 0.4 ± 0.1, 2.0 ± 0.2, and 1.5 ± 0.7 mm, respectively. New cartilage treated with cytochalasin D was significantly thinner than new cartilage in other groups. The wet weight of untreated, cytochalasin D-treated, and double-treated new cartilage was 7.1 ± 0.6, 8.4 ± 1.0, 6.7 ± 0.4, and 10.1 ± 1.3 mg, respectively. The function weight of the dual treatment group was significantly larger than that of the other groups. The functional weight of the cytochalasin-treated group was also significantly greater than that of the untreated and hyaluronidase-treated groups. Histologically, void areas existed in the untreated, hyaluronidase treated, and double treated groups (see Figure 12). All treatments except hyaluronidase stained darker than native fetal sheep articular cartilage. Total collagen staining in all groups was less intense than that for native controls. Cytochalasin D treatment resulted in the strongest glycosaminoglycan (GAG) and total collagen staining. All constructs stained for collagen I equivalently to native fetal sheep menisci, and were particularly strongly stained around the inner diffuse zone of untreated, hyaluronidase-treated, and dual-treated neocartilage. All constructs stained minimally for collagen II. P3 biochemical and mechanical data are shown in Table 2 in Figure 14. Treatment with cytochalasin D and hyaluronidase without aggregate redifferentiation was unable to reduce collagen I production and increase collagen II production in P3 new cartilage.

In the second study of this phase, aggregate redifferentiation was introduced with cytochalasin D and hyaluronidase treatment of P3R Opt neocartilage delivered in phase 1. In P3R new cartilage (see Figure 8), P3R Opt, cytochalasin D treatment (Cyto D), and double treatment (Cyto D + Hya) resulted in structures of uniform thickness. Hyaluronidase treatment (Hya) resulted in the formation of a diffuse void region in the center of the structures. All structures except the dual treatment group were slightly bowl shaped. The diameters of untreated, cytochalasin D-treated, and double-treated structures were 8.2 ± 0.2, 6.5 ± 0.2, 5.9 ± 0.0, and 5.7 ± 0.3 mm, respectively. The structure diameter of the untreated group was significantly larger than that of the other treated groups. The structure diameter of the cytochalasin D treatment group was significantly larger than that of the hyaluronidase and dual treatment groups. The thickness of new cartilage produced by untreatment, cytochalasin D treatment, hyaluronidase treatment, and dual treatment was 0.9 ± 0.0, 0.7 ± 0.1, 0.8 ± 0.2, and 0.4 ± 0.1 mm, respectively. It was. The thickness of the double-treated new cartilage was significantly smaller than that of the other treatment groups. The wet weight of untreated, cytochalasin D-treated, and double-treated new cartilage was 50.1 ± 3.2, 28.1 ± 2.6, 23.9 ± 1.4, and 11.8 ± 3.9 mg, respectively. Histologically, diffuse void areas were present only in the hyaluronidase treatment group (see Figure 8). Glycosaminoglycan (GAG), total collagen, and collagen II staining were strongest in cytochalasin D-treated neocartilage. Biochemical and mechanical data as well as functional indices are shown in Figures 8 and 15 (Table 3). Based on the excellent functionality index, cytochalasin D treatment was selected to proceed in three stages.

Fluorescent staining of F-actin in chondrocytes showed marked differences between cell passages and treatments (see Figure 9). In P0 chondrocytes, the actin array was cortical, appearing as a ring around the periphery of each cell. Untreated P3 chondrocytes were much larger in size and showed fibrous actin arrangements within the cells similar to fibroblasts. Cytochalasin D treatment of P3 chondrocytes induced a round cell shape, and while actin was still present throughout the cell, most of it was localized to the periphery. Untreated P3R chondrocytes showed cortical actin arrays with some small fibrous regions. Cytochalasin D treatment of P3R chondrocytes localized more actin to the cortex than untreated P3R cells.

Step 3: Select P3R Opt as the optimal group in step 1, cytochalasin D treatment of P3R Opt new cartilage in step 2, and then select cytochalasin D-treated P3R Opt new cartilage in step 3. The additional effect of TCL treatment on cartilage was investigated. The new cartilage treated with cytochalasin D and TCL had a similar shape and appeared thicker than the new cartilage treated with cytochalasin D. The diameters of cytochalasin D-treated and cytochalasin D and TCL dual-treated new cartilage were 6.5 ± 0.4 and 6.4 ± 0.3 mm, respectively. The thickness of dual-treated new cartilage was significantly greater than that of cytochalasin D-treated new cartilage: 1.1 ± 0.1 and 0.8 ± 0.2 mm, respectively. The wet weight of cytochalasin D-treated and double-treated new cartilage was 87.1 ± 3.6 and 88.8 ± 1.3 mg, respectively. Histologically, the new cartilage in both groups appeared uniform (see Figure 10). Both groups stained more intensely for glycosaminoglycans (GAG) and less intensely for total collagen than native fetal articular cartilage. The dual treatment group stained more intensely for glycosaminoglycans (GAG), total collagen, and collagen II. Neither group stained for collagen I. Biochemical and mechanical data as well as functional indices are shown in Figures 10 and 16 (Table 4). The functional index showed that new cartilage treated with cytochalasin D and TCL was superior to new cartilage treated with cytochalasin D alone.

Example 4 illustrates how mimicking key salient aspects of organogenesis in vitro using purified and then highly passaged cells produces neocartilage with mechanical properties equivalent to the native articular cartilage from which the cells are derived. The gradual development of new cartilage functionality through stages 1–3 is shown in Figure 11, showing a large increase in mechanical properties. Specifically, the new cartilage aggregate elastic modulus, shear modulus, and tensile modulus were found to increase 9.6-fold, 7.2-fold, and 3.8-fold, respectively, compared to the P0 control group, and the tensile strength increased 9.0-fold. The new cartilage produced as a result of this continuous study achieved an FI of 1.42 compared to native fetal cartilage and an FI of 1.03 compared to native juvenile cartilage. This indicates that the engineered new cartilage exceeded native cell organization values for parameters measured as functional indices, indicating that adult-level properties can be achieved.

In stage 1, new cartilage from P3R cells could achieve P0 new cartilage characteristics. The functional index of P3R new cartilage seeded at optimal density was equivalent to that of P0 new cartilage seeded at optimal density. Regarding fetal sheep articular cartilage, P0 Opt achieved FI 0.77 and P3R Opt achieved FI 0.78. The process of using multi-passage cells to engineer functionally robust cellular tissues has a major translational impact as it indicates that superior new cartilage can be engineered by isolating fewer cells. Therefore, P3R Opt new cartilage was delivered to subsequent steps. In the second stage, only cytochalasin D treatment was shown to be necessary to produce excellent new cartilage from passaged/redifferentiated cells. For example, cytochalasin D treatment of P3R Opt neocartilage increased compressive stiffness by 0.9-fold, tensile stiffness by 1.0-fold, and tensile strength by 2.7-fold, resulting in an FI of 1.1 for native fetal cartilage and native juvenile cartilage. 0.83 was calculated. Therefore, cytochalasin D treatment of P3R Opt new cartilage was delivered. The addition of TCL treatment in step 3 was shown to promote cross-linking-based maturation and improve the functional properties of neocartilage to achieve FI 1.42 for native fetal cartilage and FI 1.03 for native juvenile cartilage. The aggregate modulus was higher than native fetal cartilage and the tensile modulus was within the range of native levels. This study represents an important step toward achieving biomimetic articular cartilage and doing so using multi-passage cells.

In stage 1, P3R Opt new cartilage achieved FI equivalent to P0 Opt new cartilage. The functional properties of the construct within P0 new cartilage increased with increasing seeding density until a plateau was reached. However, in P3R new cartilage, functional properties decreased as seeding density increased. This contrasts with traditional tissue engineering strategies, which assume that primary cells are synthetically more competent than passaged cells and that large numbers of cells are required to produce good de novo tissue. In this study, chondrocytes were expanded more than 4,000-fold and seeded at a lower density than required for primary cells to achieve new cartilage with larger diameter and equivalent FI. The aggregate modulus, glycosaminoglycan (GAG)/DNA production, and collagen/DNA production of P3R Opt new cartilage were 0.3, 2.2, and 2.6 times greater, respectively, than those of P0 Opt new cartilage (see Figure 6). Although the collagen content of P3R Opt new cartilage was equivalent to that of P0 Opt new cartilage, the tensile stiffness and strength of P3R Opt new cartilage were significantly reduced. Considering the importance of collagen cross-linking for the tensile properties of cartilage, the reduced pyridinoline content of P3R Opt new cartilage is likely the reason for this, as addressed by three steps of TCL treatment. By optimizing self-assembly culture conditions as well as chondrogenically controlled expansion and aggregate redifferentiation methods, it was possible to engineer robust new cartilage from multiply passaged cells. Achieving this required 8,000 times fewer primary cells than engineering new cartilage from unpassaged cells.

It was unexpected that by examining different seeding densities across passage conditions, passaged chondrocytes could be reprogrammed to exhibit more immature behavior. At cell seeding densities of 2, 3, and 4 million, P3R chondrocytes were more synthetic than P0 chondrocytes. Example 4 mimics developmentally occurring proliferation, condensation, differentiation, and tissue formation with in vitro steps such as monolayer expansion, aggregate redifferentiation, and self-assembly. Evidence suggests that this reprograms P3R chondrocytes to a more immature state, allowing for increased production of matrix molecules. For example, the matrix secreted by P3R chondrocytes better reflected the composition of the articular cartilage extracellular matrix (ECM) in the early stages. As native cartilage matures, collagen VI staining, which is present throughout the extracellular matrix (ECM), becomes confined to the pericellular matrix and collagen II staining increases. The pyridinoline content in native cartilage also increases significantly over a long period of time as cartilage matures. In this study, P3R new cartilage had lower collagen II staining intensity, higher collagen VI staining intensity, and lower pyridinoline levels compared to P0 new cartilage (see Figures 6 and 7). These data support the claim that P3R chondrocytes are in a more immature state than P0 chondrocytes.

A culture technique called macromolecular crowding has been used to enhance cartilage matrix production and maturation by chondrocytes in monolayers, but shows negative effects in 3D cultures. When Ficoll 70 and Ficoll 40 were applied to monolayer chondrocytes, collagen II expression and production of glycosaminoglycan (GAG) and total collagen increased. However, in the 3D pellet culture model, macromolecular congestion treatment resulted in cartilage matrix deterioration starting from the second day of culture. The pro-inflammatory cytokine IL-6 was also detected in the medium of high-density cultures but not in low-density cultures. This study showed that P3R chondrocytes have the potential to be highly synthetic. It is known that matrix deposition in self-assembled new cartilage begins 1 day after cell seeding.

Passaged chondrocytes display a strong chondrogenic phenotype in self-assembled new cartilage. Phenotypic validation of redifferentiated chondrocytes and chondrocytes in culture using the newly proposed mechanism of self-induced macromolecular crowding and inflammatory cytokine-regulated matrix production is needed. In osteoarthritis, chondrocytes exhibit proliferation, increased synthesis of matrix molecules including collagen X, hypertrophy, and mineralization. To verify the chondrogenic phenotype of P3R new cartilage, P3R new cartilage and P0 new cartilage were stained with collagen VI, collagen X, alizarin red, and Von Kossa (see Figure 7). P3R new cartilage stained more intensely for collagen VI than P0 new cartilage. All groups stained weakly for collagen Neither group stained with alizarin red or Von Kossa, indicating no mineralization. In addition to the large voids observed in P3R new cartilage, the presence of collagen Additionally, the size of the lumen is reduced in high-density P3R new cartilage. Since collagen VI, collagen

Cytochalasin D treatment of passaged/redifferentiated cells in step 2 further enhanced the chondrogenic phenotype. Cytochalasin D treatment of P3R Opt new cartilage (delivered in stage 1) increased the aggregate modulus by 0.9-fold compared to the untreated group (see Figure 8) and increased it to the level of native articular cartilage in adult sheep. Tensile stiffness and strength also increased by 1.0- and 2.7-fold, respectively, due to minor concomitant increases in collagen and pyridinoline content. This treatment produced new cartilage with an FI of 1.1 for native fetal cartilage and 0.83 for native juvenile cartilage. The success of this treatment on passaged/redifferentiated cells motivated studies of its effects on passaged non-redifferentiated (P3) chondrocytes. Cytochalasin D treatment of P3 new cartilage was the only one of all treatments that resulted in a flat and uniform structure (see Figure 12). However, as can be seen from the strong collagen I staining and low biochemical content and mechanical properties (see Figure 14 (Table 2)), this action alone was not sufficient to affect redifferentiation enough to reveal changes in the functional properties of the construct. For P3 neocartilage, functional indices were not calculated for any treatment because the constructs were not testable in tension and morphologically unacceptable. The mode of action of cytochalasin D was confirmed by visualizing actin in P3R and P3 chondrocytes, similar to that observed in P0 cells (see Figure 9). Cytochalasin D treatment significantly improved the cortical organization of F-actin in P3 and P3R chondrocytes.

The third stage mimicked the progression of cell tissue formation by enhancing neocartilage matrix deposition and cross-linking to achieve a unique level of tensile properties. In stage 1, the pyridinoline/water weight of P3R new cartilage was significantly reduced compared to P0 new cartilage. These levels remained consistently low in Phase 2. Although this prevents the neocartilage from achieving improved tensile properties, the later development of pyridinoline cross-links compared to other matrix components mimics native cartilage maturation. TCL treatment, which has been shown to increase collagen content and cross-linking within the collagen network, was applied in three stages. This treatment actually increased collagen/water weight by 0.9 times and pyridinoline/water weight by 2.9 times, as well as increased tensile stiffness by 1.7 times and tensile strength by 3.5 times without changing compressive stiffness. These tensile properties are within the range reported for juvenile sheep articular cartilage. TCL treated neocartilage also achieved FI of 1.42 against native fetal cartilage and 1.03 against native juvenile cartilage. This indicates that the properties of the engineered new cartilage in this study are now approaching adult levels. Mimicking the key steps of native cartilage formation and following a developmentally inspired sequence of matrix development and maturation enabled purified passaged/redifferentiated cell neocartilage to achieve tensile properties in the range of native cartilage.

By mimicking key aspects of native cartilage formation and applying developmentally inspired chemical stimuli, this study was able to engineer neocartilage from cells expanded more than 4,000-fold with functional properties approaching native adult cartilage. In addition to chondrogenically coordinated expansion, aggregate redifferentiation, and optimized self-assembly of neocartilage, treatment with ACK lysis buffer, cytochalasin D, and TCL resulted in mature neocartilage with the highest functional index reported by our group. This was created. It was also confirmed that the passaged cells could be reconditioned to a more synthetic state. Mechanisms based on self-induced macromolecular congestion and cytokine-regulated feedback inhibition of cartilage matrix synthesis in high-density 3D cultures provide a plausible explanation for seeding density-dependent matrix synthesis. Finally, updated functionality indices are provided that account for the importance of cell-tissue cross-linking. This study takes a step toward establishing a protocol to generate native-like engineered new cartilage from 8,000 times fewer primary cells than previous methods.

Example 5 - Shearing

Example 5 describes a method of using shear to select cells based on membrane properties. Example 5 shows a protocol for purifying articular chondrocytes by applying shear.

Cell Isolation: Fetal sheep ACs were isolated from the femoral condyles and trochlear grooves of Dorper crossbred sheep at 120-125 days of gestation (UC Davis School of Veterinary Medicine). Minced cartilage tissue was washed with PBS and incubated with 500 units/mL collagenase in chondrogenesis medium + 3% (v/v) FBS (Atlanta Biologicals, Lawrenceville, GA) for 18 h at 37°C/10% CO2. ) digested with type II (Worthington Biochemical, Lakewood, NJ). The cells are then filtered through a 70 μm filter, washed with washing medium, and counted.

Protocol to introduce shear to purify chondrocytes: (1) Place approximately 50 mL of cell solution into a Petri dish. (2) Immerse the paddle rotor in the Petri dish and rotate it at 20 rpm for 3 minutes. (3) Remove the paddle rotor, wash the treated cell solution twice with washing medium, and count the remaining cells.

Example 6 - Impact/Compression

Example 6 describes a method of using shock/compression to select cells based on membrane properties. Example 6 shows a protocol for purifying articular chondrocytes by applying compression/impact.

Cell isolation: To obtain rib chondrocytes, cartilage from juvenile bovine knee joints was minced into 1-2 mm ³ pieces and cultured in 1% penicillin/streptomycin/fungizone (PSF) (BD Biosciences) and 3% for 18 h at 37°C. Digested with 0.2% type II collagenase (Worthington) in Dulbecco's modified Eagle's medium (DMEM) (Gibco) containing fetal bovine serum (Atlanta Biologicals). After digestion, chondrocytes are filtered through a 70 μm cell strainer, resuspended in colorless DMEM, and counted.

Protocol to introduce compression/impact to purify chondrocytes: (1) Place approximately 20 mL of cell solution into the conical tube. (2) Centrifuge the cell solution at 300g for 5 minutes to form a pellet. (3) Insert the relevant mesh conical trigger. Make sure the mesh size is smaller than 15 μm. Gently compress the cell pellet with a mesh trigger once every 30 s for 3 min. (4) Remove the inducer, wash the treated cell solution twice with washing medium, and count the remaining cells.

C. Purification based on membrane surface area properties

Example 7 - Stock solution

Example 7 describes a method of using stock solutions to select cells based on stiffness properties. It is not intended to limit the present invention to any particular theory or mechanism, but rather that the process of treating chondrocytes with a reservoir solution is effective in increasing the purity of viable chondrocytes by reducing the population of cells with existing undesirable stiffness properties. It appears that This treatment therefore generates a cell population enriched with viable chondrocytes without undesirable stiffness properties, thereby increasing the functional properties of the resulting self-assembled neocartilage.

Example 7 shows that native cartilage compression properties are achieved in engineered neocartilage. The invention is not limited to the methods or compositions described herein.

In Example 7, chondrocytes were isolated from the knee joint of fetal sheep, which is a highly clinically convertible cell source. First, primary (P0) fetal chondrocytes were treated with ammonium chloride-potassium chloride lysis buffer (ACK buffer) to investigate the effect on chondrocyte purity in the cell isolate and the functional properties of the resulting self-assembled new cartilage. Chondrocyte purity was assessed by cell counting. The functional properties of new cartilage were evaluated by a series of standard analyzes including compressive creep indentation, uniaxial tensile testing, glycosaminoglycan (GAG), collagen, and DNA analyses, as well as histology and IHC. Second, the seeding density of P0 and passaged redifferentiated (P3R) fetal chondrocytes was examined during the self-assembly process. Cells were seeded at 2, 3, 4, 5, and 6 million cells per 5 mm construct and the functional properties of the resulting new cartilage were assessed using the same set of assays. Finally, cytochalasin D and hyaluronidase were applied in a full-factorial design early in the self-assembly process to investigate their ability to further improve the functional properties of the resulting new cartilage. New cartilage was assessed using a standard battery of assays.

Results: Treatment of freshly isolated P0 fetal chondrocytes with ACK buffer reduced erythrocyte contamination of cell isolates by 60%. ACK treatment significantly increased 1) the aggregate elastic modulus of new cartilage by 1.8 times, 2) the shear modulus by 1.3 times, and 3) the tensile elastic modulus by 0.8 times. While delivering the ACK treatment of chondrocytes during isolation, the seeding density of P0 chondrocytes was optimized to 4 million cells per construct, further increasing the elastic modulus of new cartilage aggregates by 0.6 times and the shear modulus by 0.8 times. After passage and redifferentiation (P3R) of these cells, the seeding density was optimized to 2 million cells per construct, further increasing the aggregate modulus by 0.3-fold and the shear modulus by 0.3-fold. Application of cytochalasin D during self-assembly of ACK-treated P3R chondrocytes seeded with 2 million cells per construct significantly increased the elastic modulus of new cartilage aggregates to 400 kPa, 9.6 times that of the P0 control group.

As described above, the present invention generally provides a method of engineering cartilage with compressive properties similar to native cartilage. These methods purify isolated chondrocytes (e.g., via storage lysis buffer), optimize neocartilage seeding density, redifferentiate passaged chondrocytes through novel aggregate culture methods to ensure that primary cell neocartilage properties are preserved, and , It is characterized by promoting chondrocyte activity through cytoskeleton regulating substances.

Example 8 - Shrearing

Example 8 describes a method of using shear to select cells based on stiffness properties. Example 8 shows a protocol for purifying articular chondrocytes by applying shear. Cell isolation: Fetal sheep articular chondrocytes (foACs) were isolated from the knee joints of Dorper crossbreed sheep at 120 days of gestation. The cartilage of the condyles and trochlear grooves was minced into pieces of approximately 1 mm ³ and cultured in Dulbecco's modified Eagle's medium (DMEM containing 4.5 g/L glucose and GlutaMAX; Gibco) and 2% (v/v) penicillin/streptomycin/funzizone (PSF). ; Wash 3 times (500 G, 5 minutes) and centrifuge. Cell tissue was incubated with 0.2% (w/v) type II collagenase (Worthington) in DMEM containing 3% (v/v) fetal bovine serum (FBS; Atlanta Biologicals) for 18 h at 37°C with gentle shaking. Digest it. After digestion, the resulting cell solution is filtered through a 70 μm cell strainer.

Protocol to introduce shear to purify chondrocytes: (1) Place the cell solution into a sterile 10 mL syringe. (2) Attach the syringe to a microfluidic device with a 75 μm - 200 μm diameter channel. (3) Slowly press the syringe plunger to allow the cell solution to flow through the microfluidic device and into the conical tube reservoir. (4) When the syringe is fully depressed, inject another 20 mL of DMEM into the microfluidic device. (5) Wash the treated cell solution twice with washing medium and count the remaining cells.

Example 9 - Impact/Compression

Example 9 describes a method of using impact/compression to select cells based on stiffness properties. Example 9 shows a protocol for purifying articular chondrocytes by applying compression/impact. Cell isolation: Juvenile bovine articular chondrocytes are harvested from the patellofemoral surface of bovine knee joints. Articular cartilage was minced to approximately 1 mm ³ and cultured in Dulbecco's modified Eagle's medium (DMEM containing 4.5 g/L glucose and GlutaMAX; Gibco) and 2% (v/v) penicillin/streptomycin/fungizone (PSF; BD Biosciences). ) and centrifuged 3 times (500 G, 5 minutes). Finely chopped tissue was incubated with 0.2% (w/v) type II collagenase (collagenase, Worthington) in DMEM containing 3% (v/v) fetal bovine serum (FBS; Atlanta Biologicals) for 18 hours at 37°C. Let it digest for a while. After digestion, the resulting cell solution was filtered through a 70 μm cell strainer, centrifuged (500 G, 5 minutes), and resuspended in colorless DMEM.

Protocol to introduce shock/compression to purify chondrocytes: (1) Place approximately 50 mL of cell solution into a Petri dish. Place 20 glass beads of 0.25 - 0.5 mm in a Petri dish. (2) Immerse the paddle rotor in the Petri dish and rotate it at 20 rpm for 3 minutes. (3) Remove the paddle rotor and pipette the cell solution into the conical tube. (4) Wash the glass beads with 50 mL DMEM and add the washing solution to the conical tube containing the treated cell solution. (5) Wash the treated cell solution twice with washing medium and count the remaining cells.

Example 10

Example 10 illustrates a process for engineering native-like neocartilage by improving the applicability of purified and expanded chondrocytes. Additionally, the present invention is not limited to the methods or compositions described herein.

Chondrocytes were isolated from fetal sheep knee joints, and fetal cells represent a highly clinically relevant cell type for tissue engineering. First, ACK buffer treatment of primary (P0) chondrocytes reduced red blood cell contamination by 60% and increased the new cartilage aggregate modulus (1.8-fold), shear modulus (1.3-fold), and tensile modulus (0.8-fold). Afterwards, the seeding density of expansion/redifferentiation (P3R) chondrocytes was optimized to 2 million cells per construct to further increase the aggregate modulus (1.0-fold) and shear modulus (1.1-fold). Finally, cytochalasin D treatment further increased the elastic modulus of the new cartilage aggregate to 400 kPa, equivalent to native cartilage and 9.6 times that of the untreated P0 control group. P3R cells treated with ACK buffer and cytochalasin D specifically produced new cartilage with compression properties that were superior to P0 new cartilage and similar to native cartilage. Through these sequential studies, it was possible to engineer robust new cartilage using 4,000 times fewer primary cells, and specifically, using 1,000 primary cells per P3R construct compared to 4,000,000 per P0 construct, to produce expanded cartilage for tissue engineering. The clinical applicability of chondrocytes has been greatly improved.

Example 11

Example 11 describes cell isolation and processing methods. The invention is not limited to the methods and compositions described below. For example, the present invention is not limited to collecting chondrocytes from rib cartilage tissue, and the present invention is not limited to ACK buffer concentration, etc. As previously discussed, chondrocytes can be harvested from other cartilage tissues.

Isolate samples from human rib cartilage tissue. The rib cartilage tissue is dissected by removing the muscle, fatty tissue, and perichondrium, leaving essentially only the cartilage. The harvested rib cartilage is further dissected into smaller pieces, which are then centrifuged and washed. Next, the costal cartilage pieces were subjected to a digestion step using enzymes to obtain a single cell suspension, for example, the costal cartilage pieces were incubated with enzymes for a period of time while gently shaking.

The cell suspension is then processed into stock solution. For example, the cell suspension is treated with pre-warmed ACK buffer containing ammonium chloride, potassium bicarbonate and EDTA tetrasodium salt. In some embodiments, the component concentrations of the ACK buffer include, but are not limited to, 154.4 mM ammonium chloride, 10 mM potassium bicarbonate, and 97.3 μM ethylenediaminetetraacetic acid (EDTA) tetrasodium salt. Next, the cell suspension is centrifuged, the supernatant is aspirated, and the pellet is resuspended in a stock solution (ACK buffer containing ammonium chloride, potassium bicarbonate, and EDTA tetrasodium salt). Next, the cell suspension is incubated with storage (ACK) buffer for a certain period of time and then centrifuged. The supernatant is aspirated and the cell pellet is washed and plated, for example for passage.

In some embodiments, the harvested rib cartilage is dissected into pieces measuring approximately 1 mm ³ . In some embodiments, the washing step includes Dulbecco's modified Eagle's medium (DMEM containing 4.5 g/L glucose and GlutaMAX; Gibco) and 2% (v/v) penicillin/streptomycin/fungizone (PSF; BD Biosciences). . In some embodiments, the digestion step includes the use of enzymes such as pronase and/or type II collagenase. In some embodiments, cell pellets are frozen instead of plated for passage.

Various modifications of the invention in addition to what is described herein will be apparent to those skilled in the art. Such modifications are intended to be included within the scope of the appended claims. Each reference cited in this application is incorporated by reference in its entirety.

Furthermore, while preferred embodiments of the invention have been shown and described, it will be readily apparent to those skilled in the art that modifications may be made without departing from the scope of the appended claims. Accordingly, the scope of the present invention is limited only by the following claims.

In some embodiments, descriptions of the invention described herein use the phrase “comprising,” which includes embodiments that may be described as “consisting of,” and thus “consisting of.” By using the phrase "which is," the description requirements for claiming one or more embodiments of the invention are met.

Example

The following examples are illustrative only and not limiting in any way.

Example 1A: A method for enriching a chondrocyte sample, comprising: (a) collecting a chondrocyte sample containing a mixed population of non-apoptotic cells and early apoptotic cells; and (b) subjecting the chondrocyte sample of (a) to processing; The method may be repeated several times alone or in combination with other treatments. Example 2A: A method of preparing a chondrocyte sample, comprising: (a) collecting a chondrocyte sample containing a mixed population of non-apoptotic early cells and early apoptotic cells; and (b) subjecting the chondrocyte sample of (a) to processing; The method may be repeated several times alone or in combination with other treatments. Example 3A: The method of Example 1A or Example 2A, wherein the treatment includes adding a stock solution. Example 4A: A method of enriching a chondrocyte sample, comprising: (a) collecting a chondrocyte sample containing a mixed population of non-apoptotic cells and early apoptotic cells; and (b) treating the chondrocyte sample of (a) with a storage solution; The method may be repeated several times alone or in combination with other treatments. Example 5A: A method of preparing a chondrocyte sample, comprising: (a) collecting a chondrocyte sample containing a mixed population of non-apoptotic early cells and early apoptotic cells; and (b) treating the chondrocyte sample of (a) with a storage solution; The method may be repeated several times alone or in combination with other treatments. Example 6A: The method of any one of Examples 3A-5A, wherein the stock solution is ammonium chloride-potassium lysis buffer (ACK buffer). Example 7A: A method of enriching a chondrocyte sample, comprising: (a) collecting a chondrocyte sample containing a mixed population of non-apoptotic cells and early apoptotic cells; and (b) treating the chondrocyte sample of (a) with ammonium chloride-potassium lysis buffer (ACK buffer); The method may be repeated several times alone or in combination with other treatments. Example 8A: A method of preparing a chondrocyte sample, comprising: (a) collecting a chondrocyte sample comprising a mixed population of non-apoptotic early cells and early apoptotic cells; and (b) treating the chondrocyte sample of (a) with ammonium chloride-potassium lysis buffer (ACK buffer); The method may be repeated several times alone or in combination with other treatments. Example 9A: The method of any one of Examples 1A-8A, wherein the treatment induces cell expansion. Example 10A: The method of any one of Examples 1A-9A, wherein the chondrocyte sample is a human chondrocyte sample. Example 11A: The method of any one of Examples 1A-10A, wherein the chondrocyte sample is a non-articular chondrocyte sample. Example 12A: The method of any one of Examples 1A-11A, wherein the cell sample is derived from a portion of a rib. Example 13A: The method of Example 12A, wherein the ribs include chondrocytes. Example 14A: The method of Example 13A, wherein the chondrocytes are non-articular chondrocytes. Example 15A: The method of any one of Examples 1A-14A, wherein the cell sample after (b) has a higher percentage of non-apoptotic early cells than before (b). Example 16A: The method of any one of Examples 1A-14A, wherein the cell sample after (b) has a lower percentage of early apoptotic cells than the pre-chondrocyte sample (b). Example 17A: The method of any one of Examples 1A-16A, wherein the cells produced after (b) are used for one or more of the following: direct use of the cells; In vitro culture of cells, including passage in a monolayer or three-dimensional environment, including suspension culture; Tissue engineering using scaffold-free systems, including self-assembly, or scaffold-based systems, including natural and synthetic materials; cell transplantation; tissue transplant; and/or transplantation. Example 18A: The method of Example 1A, further comprising (b) subsequent passage of the cells in a monolayer or three-dimensional environment. Example 19A: The method of Example 18A, further comprising producing new cartilage with the passaged cells. Example 20A: The method of any one of Examples 1A-19A, wherein (b) the cells produced or tissue engineered/produced from said cells are subjected to a treatment comprising one or more of the following: growth. factor; Cytoskeletal regulatory substances; hormone; toxic compounds; Molecules that act upstream in a signal transduction pathway; variable oxygen tension; crosslinking agent; matrix degrading enzyme, matrix molecule; and/or mechanical stimulation.

Example 1B: A method of enhancing a human chondrocyte sample, the method comprising the following steps: (a) Obtaining a human chondrocyte sample. In this case, the human chondrocyte sample includes a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) providing the human chondrocyte sample from step (a) for processing; The method can be repeated on its own or in combination with other treatments. Example 2B: A method of preparing a human chondrocyte sample, the method comprising the following steps: (a) Obtaining a human chondrocyte sample. In this case, the human chondrocyte sample includes a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) providing the human chondrocyte sample from step (a) for processing; The method can be repeated on its own or in combination with other treatments. Example 3B: The method of Example 1B or Example 2B, wherein the treatment includes adding a stock solution. Example 4B: A method of enhancing a human chondrocyte sample, the method comprising the following steps: (a) Obtaining a human chondrocyte sample. In this case, the human chondrocyte sample includes a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) treating the human chondrocyte sample from step (a) with a stock solution; The method can be repeated on its own or in combination with other treatments. Example 5B: A method of preparing a human chondrocyte sample, the method comprising the following steps: (a) Obtaining a human chondrocyte sample. In this case, the human chondrocyte sample includes a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) treating the human chondrocyte sample from step (a) with a stock solution; The method can be repeated on its own or in combination with other treatments. Example 6B: In the method of any one of Examples 3B to 5B, the storage liquid is an ACK buffer. Example 7B: A method of enhancing a human chondrocyte sample, the method comprising the following steps: (a) Obtaining a human chondrocyte sample. In this case, the human chondrocyte sample includes a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) processing the human chondrocyte sample from step (a) with a storage solution (ACK buffer); The method can be repeated on its own or in combination with other treatments. Example 8B: A method of preparing a human chondrocyte sample, the method comprising the following steps: (a) Obtaining a human chondrocyte sample. In this case, the human chondrocyte sample includes a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) processing the human chondrocyte sample from step (a) with a storage solution (ACK buffer); The method can be repeated on its own or in combination with other treatments. Example 9B: The method of any one of Examples 1B to 8B, wherein the treatment induces cell expansion. Example 10B: The method of any one of Examples 1B to 9B, wherein the human chondrocyte sample is a non-articular chondrocyte sample. Example 11B: The method of any one of Examples 1B-10B, wherein the human chondrocyte sample is from a portion of a rib. Example 12B: The method of Example 11B, wherein the chondrocytes derived from the rib region comprise non-articular chondrocytes. Example 13B: The method of any one of Examples 1B-12B, wherein the human chondrocyte sample after (b) has a higher percentage of non-pre-apoptotic cells compared to before (b). Example 14B: The method of any of Examples 1B-12B, wherein the chondrocyte sample after (b) has a lower percentage of pre-apoptotic cells compared to before (b). Example 15B: The method of any one of Examples 1B to 14B, wherein (b) the human chondrocytes produced thereafter are used for direct use of the cells; In vitro culture of cells, including subculture in a three-dimensional environment, including monolayer or suspension culture; Tissue engineering using scaffold-free systems, including self-assembly or the use of scaffold-based systems containing natural and synthetic materials; cell transplantation; tissue transplantation; and/or transplantation. Example 16B: The method of Example 1B further includes subculturing the cells in a monolayer or three-dimensional environment after (b). Example 17B: The method of Example 16B further includes the process of producing new cartilage using the subcultured cells. Example 18B: The method of any one of Examples 1B to 17B, wherein the human chondrocytes produced after (b) or the tissue engineered or prepared from the human chondrocytes are enriched with a growth factor; cytoskeleton modifier; hormone; toxic compounds; Molecules that act upstream in signaling pathways; variable oxygen tension; crosslinking agent; matrix degrading enzyme, matrix molecule; and/or mechanical stimulation.

Example 1C: A method of enhancing a non-articular chondrocyte sample, the method comprising the following steps: (a) Obtaining a non-articular chondrocyte sample. At this time, the non-articular chondrocyte sample includes a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) providing the non-articular chondrocyte sample from step (a) for processing; The method can be repeated on its own or in combination with other treatments. Example 2C: A method of preparing a non-articular chondrocyte sample, the method comprising the following steps: (a) Obtaining a non-articular chondrocyte sample. At this time, the non-articular chondrocyte sample includes a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) providing the non-articular chondrocyte sample from step (a) for processing; The method can be repeated on its own or in combination with other treatments. Example 3C: The method of Example 1C or Example 2C, wherein the treatment includes adding a stock solution. Example 4C: A method of enhancing a non-articular chondrocyte sample, the method comprising the following steps: (a) Obtaining a non-articular chondrocyte sample. At this time, the non-articular chondrocyte sample includes a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) treating the non-articular chondrocyte sample from step (a) with a stock solution; The method can be repeated on its own or in combination with other treatments. Example 5C: A method of preparing a sample of non-articular chondrocytes, the method comprising the following steps: (a) Obtaining a sample of non-articular chondrocytes. At this time, the non-articular chondrocyte sample includes a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) treating the non-articular chondrocyte sample from step (a) with a stock solution; The method can be repeated on its own or in combination with other treatments. Example 6C: In the method of any one of Examples 3C to 5C, the storage liquid is an ACK buffer. Example 7C: A method of enhancing a non-articular chondrocyte sample, the method comprising the following steps: (a) Obtaining a non-articular chondrocyte sample. At this time, the non-articular chondrocyte sample includes a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) processing the non-articular chondrocyte sample from step (a) with an ACK buffer; The method can be repeated on its own or in combination with other treatments. Example 8C: A method of preparing a non-articular chondrocyte sample, the method comprising the following steps: (a) Obtaining a non-articular chondrocyte sample. At this time, the non-articular chondrocyte sample includes a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) processing the non-articular chondrocyte sample from step (a) with an ACK buffer; The method can be repeated on its own or in combination with other treatments. Example 9C: The method of any one of Examples 1C to 8C, wherein the treatment induces cell expansion. Example 10C: The method of any one of Examples 1C to 9C, wherein the non-articular chondrocyte sample is a human non-articular chondrocyte sample. Example 11C: The method of any of Examples 1C-10C, wherein the non-articular chondrocyte sample is from a portion of a rib. Example 12C: The method of any of Examples 1C-11C, wherein the non-articular chondrocyte sample after (b) has a higher percentage of non-pre-apoptotic cells compared to before (b). Example 13C: The method of any of Examples 1C-11C, wherein the non-articular chondrocyte sample after (b) has a lower percentage of pre-apoptotic cells compared to before (b). Example 14C: The method of any one of Examples 1C to 13C, wherein (b) the non-articular chondrocytes produced thereafter are used for direct use of the cells; In vitro culture of cells, including subculture in a three-dimensional environment, including monolayer or suspension culture; Tissue engineering using scaffold-free systems, including self-assembly or the use of scaffold-based systems comprising natural and synthetic materials; cell transplantation; tissue transplantation; and/or transplantation. Example 15C: The method of Example 1C further includes subculturing the cells in a monolayer or three-dimensional environment after (b). Example 16C: The method of Example 15C further includes the process of producing new cartilage using the subcultured cells. Example 17C: The method of any one of Examples 1C to 16C, wherein the non-articular chondrocytes produced after (b) or the tissue engineered or prepared from the non-articular chondrocytes are enriched with a growth factor; cytoskeleton modifier; hormone; toxic compounds; Molecules that act upstream in signaling pathways; variable oxygen tension; crosslinking agent; matrix degrading enzyme, matrix molecule; and/or mechanical stimulation.

Example 1D: A method of improving a cell sample derived from a portion of a rib, the method comprising the following steps: (a) Obtaining a cell sample derived from a portion of the rib. wherein the cell sample derived from the portion of the rib contains a mixed population of non-pre-apoptotic and pre-apoptotic cells; and (b) subjecting a cell sample derived from the portion of the rib from step (a) to processing; The method can be repeated on its own or in combination with other treatments. Example 2D: A method of preparing a cell sample from a portion of a rib, the method comprising the following steps: (a) Obtaining a cell sample from a portion of the rib. wherein the cell sample derived from the portion of the rib contains a mixed population of non-pre-apoptotic and pre-apoptotic cells; and (b) subjecting a cell sample derived from the portion of the rib from step (a) to processing; The method can be repeated on its own or in combination with other treatments. Example 3D: The method of Example 1D or Example 2D, wherein the treatment includes adding a stock solution. Example 4D: A method of improving a cell sample derived from a portion of a rib, the method comprising the following steps: (a) Obtaining a cell sample derived from a portion of the rib. wherein the cell sample derived from the portion of the rib contains a mixed population of non-pre-apoptotic and pre-apoptotic cells; and (b) treating a cell sample derived from a portion of the rib from step (a) with a stock solution; The method can be repeated on its own or in combination with other treatments. Example 5D: A method of preparing a cell sample from a portion of a rib, the method comprising the following steps: (a) Obtaining a cell sample from a portion of the rib. wherein the cell sample derived from the portion of the rib contains a mixed population of non-pre-apoptotic and pre-apoptotic cells; and (b) treating a cell sample derived from a portion of the rib from step (a) with a stock solution; The method can be repeated on its own or in combination with other treatments. Example 6D: In the method of any one of Examples 3D to 5D, the storage liquid is an ACK buffer. Example 7D: A method of improving a cell sample derived from a portion of a rib, the method comprising the following steps: (a) Obtaining a cell sample derived from a portion of the rib. wherein the cell sample derived from the portion of the rib contains a mixed population of non-pre-apoptotic and pre-apoptotic cells; and (b) processing the cell sample derived from a portion of the rib from step (a) with a storage solution (ACK buffer); The method can be repeated on its own or in combination with other treatments. Example 8D: A method of preparing a cell sample from a portion of a rib, the method comprising the following steps: (a) Obtaining a cell sample from a portion of the rib. wherein the cell sample derived from the portion of the rib contains a mixed population of non-pre-apoptotic and pre-apoptotic cells; and (b) processing the cell sample derived from a portion of the rib from step (a) with a storage solution (ACK buffer); The method can be repeated on its own or in combination with other treatments. Example 9D: The method of any one of Examples 1D to 8D, wherein the treatment induces cell expansion. Example 10D: The method of any of Examples 1D-9D, wherein the cell sample derived from a portion of the rib is a chondrocyte. Example 11D: The method of any of Examples 1D-10D, wherein the cell sample derived from a portion of the rib is non-articular chondrocytes. Example 12D: The method of any one of Examples 1D-11D, wherein the cell sample derived from a portion of the rib is a human cell. Example 13D: The method of any one of Examples 1D to 12D, wherein the portion of the rib includes chondrocytes. Example 14D: The method of Example 13D, wherein the chondrocytes are non-articular chondrocytes. Example 15D: The method of any of Examples 1D-14D, wherein the cell sample derived from a portion of the rib after (b) contains a higher percentage of non-pre-apoptotic cells compared to before (b) have Example 16D: The method of any one of Examples 1D-14D, wherein the cell sample derived from a portion of the rib after (b) has a lower percentage of pre-apoptotic cells compared to before (b). Example 17D: The method of any one of Examples 1D to 16D, wherein (b) the cell sample derived from a portion of the subsequently produced rib is used for direct use of the cells; In vitro culture of cells, including subculture in a three-dimensional environment, including monolayer or suspension culture; Tissue engineering using scaffold-free systems, including self-assembly or the use of scaffold-based systems containing natural and synthetic materials; cell transplantation; tissue transplantation; and/or transplantation. Example 18D: The method of Example 1D further includes subculturing the cells in a monolayer or three-dimensional environment after (b). Example 19D: The method of Example 18D further includes the process of producing new cartilage using the subcultured cells. Example 20D: The method of any one of Examples 1D to 19D, wherein (b) the sample of cells derived from a portion of the subsequently produced rib or tissue engineered or prepared from the cells includes a growth factor; cytoskeleton modifier; hormone; toxic compounds; Molecules that act upstream in signaling pathways; variable oxygen tension; crosslinking agent; matrix degrading enzyme, matrix molecule; and/or mechanical stimulation.

Example 1E: A method of enhancing a human non-articular chondrocyte sample, the method comprising the following steps: (a) Obtaining a human non-articular chondrocyte sample. wherein the human non-articular chondrocyte sample includes a mixed population of non-pre-apoptotic and pre-apoptotic chondrocytes; and (b) subjecting the human non-articular chondrocyte sample from step (a) to processing; The method can be repeated on its own or in combination with other treatments. Example 2E: A method of preparing a human non-articular chondrocyte sample, the method comprising the following steps: (a) Obtaining a human non-articular chondrocyte sample. wherein the human non-articular chondrocyte sample includes a mixed population of non-pre-apoptotic and pre-apoptotic chondrocytes; and (b) subjecting the human non-articular chondrocyte sample from step (a) to processing; The method can be repeated on its own or in combination with other treatments. Example 3E: The method of Example 1E or Example 2E, wherein the treatment includes adding a stock solution. Example 4E: A method of enhancing a human non-articular chondrocyte sample, the method comprising the following steps: (a) Obtaining a human non-articular chondrocyte sample. wherein the human non-articular chondrocyte sample includes a mixed population of non-pre-apoptotic and pre-apoptotic chondrocytes; and (b) treating the human non-articular chondrocyte sample from step (a) with a stock solution; The method can be repeated on its own or in combination with other treatments. Example 5E: A method of preparing a human non-articular chondrocyte sample, the method comprising the following steps: (a) Obtaining a human non-articular chondrocyte sample. wherein the human non-articular chondrocyte sample includes a mixed population of non-pre-apoptotic and pre-apoptotic chondrocytes; and (b) treating the human non-articular chondrocyte sample from step (a) with a stock solution; The method can be repeated on its own or in combination with other treatments. Example 6E: In the method of any one of Examples 3E to 5E, the storage liquid is an ACK buffer. Example 7E: A method of enhancing a human non-articular chondrocyte sample, the method comprising the following steps: (a) Obtaining a human non-articular chondrocyte sample. wherein the human non-articular chondrocyte sample includes a mixed population of non-pre-apoptotic and pre-apoptotic chondrocytes; and (b) processing the human non-articular chondrocyte sample from step (a) with an ACK buffer; The method can be repeated on its own or in combination with other treatments. Example 8E: A method of preparing a human non-articular chondrocyte sample, the method comprising the following steps: (a) Obtaining a human non-articular chondrocyte sample. wherein the human non-articular chondrocyte sample includes a mixed population of non-pre-apoptotic and pre-apoptotic chondrocytes; and (b) processing the human non-articular chondrocyte sample from step (a) with an ACK buffer; The method can be repeated on its own or in combination with other treatments. Example 9E: The method of any one of Examples 1E to 8E, wherein the treatment induces cell expansion. Example 10E: The method of any of Examples 1E-9E, wherein the human non-articular chondrocyte sample is from a portion of a rib. Example 11E: The method of any one of Examples 1E-10E, wherein the human non-articular chondrocyte sample after (b) has a higher percentage of non-pre-apoptotic cells compared to before (b) . Example 12E: The method of any one of Examples 1E-10E, wherein the human non-articular chondrocyte sample after (b) has a lower percentage of pre-apoptotic cells compared to before (b). Example 13E: The method of any one of Examples 1E-12E, wherein (b) the human non-articular chondrocyte sample produced thereafter is subjected to direct use of the cells; In vitro culture of cells, including subculture in a three-dimensional environment, including monolayer or suspension culture; Tissue engineering using scaffold-free systems, including self-assembly or the use of scaffold-based systems comprising natural and synthetic materials; cell transplantation; tissue transplantation; and/or transplantation. Example 14E: The method of Example 1E further includes subculturing the cells in a monolayer or three-dimensional environment after (b). Example 15E: The method of Example 14E further includes the process of producing new cartilage using the subcultured cells. Example 16E: The method of any one of Examples 1E-15E, wherein (b) the human non-articular chondrocyte sample produced or the tissue engineered or prepared from the cells is enriched with a growth factor; cytoskeleton modifier; hormone; toxic compounds; Molecules that act upstream in signaling pathways; variable oxygen tension; crosslinking agent; matrix degrading enzyme, matrix molecule; and/or mechanical stimulation.

Example 1F: A method of improving a human cell sample derived from a portion of a rib, the method comprising the following steps: (a) Obtaining a human cell sample derived from a portion of the rib. wherein the human cell sample derived from the portion of the rib contains a mixed population of non-pre-apoptotic and pre-apoptotic cells; and (b) subjecting the human cell sample derived from the portion of the rib from step (a) to processing; The method can be repeated on its own or in combination with other treatments. Example 2F: A method of preparing a human cell sample derived from a portion of a rib, the method comprising the following steps: (a) Obtaining a human cell sample derived from a portion of the rib. wherein the human cell sample derived from the portion of the rib contains a mixed population of non-pre-apoptotic and pre-apoptotic cells; and (b) subjecting the human cell sample derived from the portion of the rib from step (a) to processing; The method can be repeated on its own or in combination with other treatments. Example 3F: The method of Example 1F or Example 2F, wherein the treatment includes adding a stock solution. Example 4F: A method of improving a human cell sample derived from a portion of a rib, the method comprising the following steps: (a) Obtaining a human cell sample derived from a portion of the rib. wherein the human cell sample derived from the portion of the rib contains a mixed population of non-pre-apoptotic and pre-apoptotic cells; and (b) treating the human cell sample derived from a portion of the rib from step (a) with a stock solution; The method can be repeated on its own or in combination with other treatments. Example 5F: A method of preparing a human cell sample derived from a portion of a rib, the method comprising the following steps: (a) Obtaining a human cell sample derived from a portion of the rib. wherein the human cell sample derived from the portion of the rib contains a mixed population of non-pre-apoptotic and pre-apoptotic cells; and (b) treating the human cell sample derived from a portion of the rib from step (a) with a stock solution; The method can be repeated on its own or in combination with other treatments. Example 6F: In the method of any one of Examples 3F to 5F, the storage liquid is an ACK buffer. Example 7F: A method of improving a human cell sample derived from a portion of a rib, the method comprising the following steps: (a) Obtaining a human cell sample derived from a portion of the rib. wherein the human cell sample derived from the portion of the rib contains a mixed population of non-pre-apoptotic and pre-apoptotic cells; and (b) processing the human cell sample derived from a portion of the rib from step (a) with a storage solution (ACK buffer); The method can be repeated on its own or in combination with other treatments. Example 8F: A method of preparing a human cell sample derived from a portion of a rib, the method comprising the following steps: (a) Obtaining a human cell sample derived from a portion of the rib. wherein the human cell sample derived from the portion of the rib contains a mixed population of non-pre-apoptotic and pre-apoptotic cells; and (b) processing the human cell sample derived from a portion of the rib from step (a) with a storage solution (ACK buffer); The method can be repeated on its own or in combination with other treatments. Example 9F: The method of any one of Examples 1F to 8F, wherein the treatment induces cell expansion. Example 10F: The method of any one of Examples 1F to 9F, wherein the human cell sample derived from a portion of the rib is a chondrocyte. Example 11F: The method of any one of Examples 1F-10F, wherein the human cell sample derived from a portion of the rib is a non-articular chondrocyte. Example 12F: The method of any one of Examples 1F to 11F, wherein the portion of the rib includes chondrocytes. Example 13F: The method of Example 12F, wherein the chondrocytes are non-articular chondrocytes. Example 14F: The method of any one of Examples 1F-13F, wherein the human cell sample derived from a portion of the rib after (b) has a higher percentage of non-pre-apoptotic cells compared to before (b) has Example 15F: The method of any one of Examples 1F-13F, wherein the human cell sample derived from a portion of the rib after (b) has a lower percentage of pre-apoptotic cells compared to before (b) . Example 16F: The method of any one of Examples 1F to 15F, wherein (b) the human cell sample derived from a portion of the subsequently produced rib is subjected to direct use of the cells; In vitro culture of cells, including subculture in a three-dimensional environment, including monolayer or suspension culture; Tissue engineering using scaffold-free systems, including self-assembly or the use of scaffold-based systems containing natural and synthetic materials; cell transplantation; tissue transplantation; and/or transplantation. Example 17F: The method of Example 1F further includes the step of subculturing cells in a monolayer or three-dimensional environment after (b). Example 18F: The method of Example 17F further includes the process of producing new cartilage using the subcultured cells. Example 19F: The method of any one of Examples 1F to 18F, wherein (b) the sample of cells derived from a portion of the subsequently produced rib or tissue engineered or prepared from the cells comprises a growth factor; cytoskeleton modifier; hormone; toxic compounds; Molecules that act upstream in signaling pathways; variable oxygen tension; crosslinking agent; matrix degrading enzyme, matrix molecule; and/or mechanical stimulation.

Claims (54)

As a method of strengthening a chondrocyte sample,
a) obtaining the chondrocyte sample from cartilage tissue; and
b) treating the chondrocyte sample of (a) with a hypotonic solution. Method for strengthening a chondrocyte sample comprising a. According to paragraph 1,
The method of strengthening a chondrocyte sample wherein the storage solution is ammonium chloride potassium hemolysis buffer (ACK buffer). As a method of strengthening a chondrocyte sample,
a) obtaining the chondrocyte sample from cartilage tissue; and
b) treating the chondrocyte sample of (a) with a hypotonic solution,
The method of strengthening a chondrocyte sample wherein the storage solution is ammonium chloride potassium hemolysis buffer (ACK buffer). According to any one of claims 1 to 3,
The processing step is a method of strengthening a chondrocyte sample to induce cell expansion. According to any one of claims 1 to 4,
The chondrocyte sample is a method of strengthening a chondrocyte sample, wherein the chondrocyte sample is a human-derived cartilage cell. According to any one of claims 1 to 5,
The chondrocyte sample is a method of strengthening a chondrocyte sample derived from a portion of the rib. According to any one of claims 1 to 6,
A method of strengthening a chondrocyte sample, wherein the chondrocyte sample is a sample derived from non-articular chondrocytes. According to any one of claims 1 to 7,
A method of enriching a chondrocyte sample, wherein the chondrocyte sample includes a mixed population of non-pre-apoptotic cells and pre-apoptotic cells. According to clause 8,
A method of enriching a chondrocyte sample after (b), wherein the chondrocyte sample has a higher proportion of non-pre-apoptotic cells compared to before (b). According to clause 8,
A method of enriching a chondrocyte sample after (b) in which the chondrocyte sample has a lower percentage of pre-apoptotic cells compared to before (b). According to any one of claims 1 to 10,
The chondrocytes produced after (b) may be used directly; In vitro culture of cells, including subculture in a three-dimensional environment, including monolayer or suspension culture; Tissue engineering using scaffold-free systems, including self-assembly or the use of scaffold-based systems containing natural and synthetic materials; cell transplantation; tissue transplantation; and/or transplantation. In the method of claim 1 or 3,
After (b), the method for strengthening a chondrocyte sample further includes subculturing the cells in a single layer or three-dimensional environment. According to clause 12,
A method of strengthening a chondrocyte sample further comprising producing new cartilage using the subcultured cells. According to any one of claims 1 to 13,
The chondrocytes produced after (b) or the tissue engineered or manufactured from the chondrocytes produced after (b) include growth factors; cytoskeleton modifier; hormone; toxic compounds; Molecules that act upstream in signaling pathways; variable oxygen tension; crosslinking agent; matrix degrading enzyme, matrix molecule; and/or a method of strengthening a chondrocyte sample that has undergone a process including at least one from the group consisting of mechanical stimulation. As a method for strengthening human chondrocyte samples,
a) obtaining the human chondrocyte sample; and
b) treating the human chondrocyte sample of (a) with a hypotonic solution,
The method is a method of enriching human chondrocyte samples that can be repeated alone or in combination with other treatments. According to clause 15,
The method of strengthening a human chondrocyte sample wherein the storage solution is ammonium chloride potassium hemolysis buffer (ACK buffer). As a method for strengthening human chondrocyte samples,
a) obtaining the human chondrocyte sample from cartilage tissue; and
b) treating the human chondrocyte sample of (a) with a hypotonic solution, wherein the hypotonic solution is ammonium chloride potassium hemolysis buffer (ACK buffer);
The method of strengthening a human chondrocyte sample is a method of strengthening a human chondrocyte sample that can be repeated alone or in combination with other treatments. According to any one of claims 15 to 17,
A method of enriching a human chondrocyte sample, wherein the human chondrocyte sample is a sample of non-articular chondrocyte. According to any one of claims 15 to 18,
A method of strengthening a human chondrocyte sample, wherein the human chondrocyte sample is derived from a portion of a rib. According to any one of claims 15 to 19,
A method of strengthening a human chondrocyte sample, wherein the treatment induces cell expansion. According to any one of claims 16 to 20,
A method of enriching a human chondrocyte sample, wherein the human chondrocyte sample includes a mixed population of non-pre-apoptotic cells and pre-apoptotic cells. According to clause 21,
A method of enriching a human chondrocyte sample after (b), wherein the human chondrocyte sample has a higher proportion of non-pre-apoptotic cells compared to before (b). According to clause 21,
A method of enriching a human chondrocyte sample after (b), wherein the human chondrocyte sample has a lower percentage of pre-apoptotic cells compared to before (b). According to any one of claims 15 to 23,
The human chondrocytes produced after (b) may be used directly; In vitro culture of cells, including subculture in a three-dimensional environment, including monolayer or suspension culture; Tissue engineering using scaffold-free systems, including self-assembly or the use of scaffold-based systems containing natural and synthetic materials; cell transplantation; tissue transplantation; and/or transplantation. According to claim 15 or 17,
After (b), the method for strengthening a human chondrocyte sample further includes subculturing the cells in a single layer or three-dimensional environment. According to clause 25,
A method of strengthening a human chondrocyte sample further comprising producing new cartilage using the subcultured cells. According to any one of claims 15 to 26,
The chondrocytes produced after (b) or the tissue engineered or manufactured from the chondrocytes produced after (b) include growth factors; cytoskeleton modifier; hormone; toxic compounds; Molecules that act upstream in signaling pathways; variable oxygen tension; crosslinking agent; matrix degrading enzyme, matrix molecule; and/or mechanical stimulation. As a method of strengthening a non-articular chondrocyte sample,
a) obtaining the non-articular chondrocyte sample from cartilaginous tissue; and
b) treating the non-articular chondrocyte sample of (a) with a storage solution. A method of strengthening a non-articular chondrocyte sample comprising a. According to clause 28,
The method of strengthening a non-articular chondrocyte sample, wherein the stock solution is ammonium chloride potassium hemolysis buffer (ACK buffer). As a method of strengthening a non-articular chondrocyte sample,
a) obtaining the non-articular chondrocyte sample from cartilaginous tissue; and
b) treating the non-articular chondrocyte sample of (a) with a hypotonic solution, wherein the hypotonic solution is ammonium chloride potassium hemolysis buffer (ACK buffer); non-articular chondrocytes comprising How to strengthen the sample. According to any one of claims 28 to 30,
A method of enriching a non-articular chondrocyte sample, wherein the non-articular chondrocyte sample is a sample of human non-articular chondrocytes. According to any one of claims 28 to 31,
A method of enriching a non-articular chondrocyte sample, wherein the non-articular chondrocyte sample is derived from a portion of a rib. According to any one of claims 28 to 32,
A method of strengthening a non-articular chondrocyte sample wherein the treatment induces cell expansion. According to any one of claims 28 to 33,
A method of enriching a non-articular chondrocyte sample, wherein the non-articular chondrocyte sample comprises a mixed population of non-pre-apoptotic cells and pre-apoptotic cells. In the method of paragraph 34,
A method of enriching a non-articular chondrocyte sample after (b), wherein the non-articular chondrocyte sample has a higher proportion of non-pre-apoptotic cells compared to before (b). In the method of paragraph 34,
A method of enriching a non-articular chondrocyte sample after (b), wherein the non-articular chondrocyte sample has a lower percentage of pre-apoptotic cells compared to before (b). According to any one of claims 28 to 36,
The non-articular chondrocytes produced after (b) include direct use of the cells; In vitro culture of cells, including subculture in a three-dimensional environment, including monolayer or suspension culture; Tissue engineering using scaffold-free systems, including self-assembly or the use of scaffold-based systems comprising natural and synthetic materials; cell transplantation; tissue transplantation; and/or transplantation. According to any one of paragraphs 28 or 30,
After (b), the method for strengthening a non-articular chondrocyte sample further includes subculturing the cells in a single layer or three-dimensional environment. In the method of paragraph 38,
A method of strengthening a non-articular chondrocyte sample further comprising producing new cartilage using the subcultured cells. According to any one of claims 28 to 39,
The non-articular chondrocytes produced after (b) or the tissue engineered or manufactured from the non-articular chondrocytes produced after (b) may contain growth factors; cytoskeleton modifier; hormone; toxic compounds; Molecules that act upstream in signaling pathways; variable oxygen tension; crosslinking agent; matrix degrading enzyme, matrix molecule; and/or mechanical stimulation. A method for producing a human chondrocyte sample, comprising:
a) obtaining a sample of human chondrocytes derived from a portion of a rib; and
b) treating the human chondrocyte sample of (a) with a hypotonic solution, wherein the hypotonic solution is ammonium chloride potassium hemolysis buffer (ACK buffer). According to clause 41,
A method of producing human cartilage cells in which a portion of the ribs includes costal cartilage tissue. According to claim 41 or 42,
A method for producing human chondrocytes, further comprising peeling a portion of the rib prior to processing the human chondrocyte sample. According to clause 43,
A method of producing human chondrocytes in which the process of peeling off a portion of the rib is a process of removing cells unsuitable for tissue formation. According to clause 43,
A method for producing human chondrocytes further comprising enzymatically digesting a portion of the rib prior to processing the human chondrocyte sample. According to any one of claims 1 to 45,
A method wherein the chondrocyte sample is a primary cell sample. As a processed cell sample produced,
a) Obtaining a cartilage sample and digesting the cartilage sample to obtain a cell suspension;
b) treating the cell suspension with a hypotonic solution,
The treated cell sample is suitable for producing new cartilage. According to clause 47,
The processed cell sample, wherein the stock solution is potassium ammonium chloride hemolysis buffer (ACK buffer). According to any one of paragraphs 47 or 48,
The processed cell sample, wherein the cartilage sample is cartilage derived from a human. According to any one of claims 47 to 49,
A treated cell sample, wherein the cartilage sample is non-articular chondrocytes. According to any one of claims 47 to 50,
The human cartilage sample is a processed cell sample derived from a portion of a rib bone. According to any one of claims 47 to 51,
The processed cell sample, wherein the cartilage sample is a primary sample. The method according to any one of claims 47 to 52,
The treated cell sample, wherein the ACK buffer contains 154.4 mM ammonium chloride, 10 mM potassium bicarbonate, and 97.3 μM EDTA tetrasodium salt. According to any one of claims 47 to 53,
The method further comprises, after (b), centrifuging the treated cell sample and resuspending the cell sample.