KR20070002028A - Tissue-like organization of cells and macroscopic tissue-like constructs, generated by macromass culture of cells, and the method of macromass culture - Google Patents

Abstract

Three-dimensional tissue-like organization of cells by high cell- seeding-density culture termed as macromass culture is described. By macromass culture, cells can be made to organize themselves into a tissue- like form without the aid of a scaffold and three-dimensional macroscopic tissue-like constructs can be made wholly from cells. Tissue-like organization and macroscopic tissue-like constructs can be generated from fibroblastic cells of mesenchymal origin (at least), which can be either differentiated cells or multipotent adult stem cells. In this work, tissue-like organization and macroscopic tissue-like constructs have been generated from dermal fibroblasts, adipose stromal cells-derived osteogenic cells, chondrocytes, and from osteoblasts. The factor causing macroscopic tissue formation is large scale culture at high cell seeding density per unit area or three-dimensional space, that is, macromass culture done on a large scale. No scaffold or extraneous matrix is used for tissue generation, the tissues are of completely cellular origin. No other agents (except high cell-seeding-density) that aid in tissue formation such as tissue-inducing chemicals, tissue-inducing growth factors, substratum with special properties, rotational culture, etc, are employed for tissue formation. These tissue-like masses have the potential for use as tissue replacements in the human body. Tissue-like organization by high cell-seeding-density macromass culture can also be generated at the microscopic level. ® KIPO & WIPO 2007

Description

TISSUE-LIKE ORGANIZATION OF CELLS AND MACROSCOPIC TISSUE-LIKE CONSTRUCTS, GENERATED BY MACROMASS CULTURE OF CELLS, produced by macromass culture of cells, AND THE METHOD OF MACROMASS CULTURE}

The present invention relates to tissue engineering. More specifically, the present invention relates to the generation of three-dimensional tissue-like tissues of cells. More specifically, the present invention relates to the fabrication of three-dimensional macroscopic, tissue-like structures for implantation possible in the human body as a treatment for diseased or damaged conditions.

The human body may suffer from several diseases or damaged conditions of other organs, one therapeutic approach for which is the exchange of damaged parts by tissue equivalents obtained or developed outpatient. For example, skin and burns or ulcers can be treated with the application of appropriate skin equivalents, unbonded gaps in fractured bones can be treated by implantation of appropriate bone substitutes, and damage to articular cartilage can be treated with appropriate cartilage. -Can be recovered by forming implants.

Every year, surgeons perform surgical procedures to treat patients who experience organ failure or tissue loss. Surgeons / physicians have been able to treat these patients by transplanting organs from one individual to another, performing reconstructive surgery, or by using mechanical devices such as kidney dialysis, artificial hip joints, or mechanical heart valves. Although this approach saved many lives, they face limitations. The limitation of transplantation of organs such as heart, liver and kidneys is not a surgical technique, but the availability of donor organs is rare.

Possible types of naturally available implants were xenografts from animals, allografts from human donors, and autografts from healthy parts of the patients themselves. Xenografts have problems with immune incompatibility and delivery of common pathogens, including retroviruses. Allografts have problems of donor's immune rejection and availability. Autografts have the problem of lack of adequate amount of adequate tissue and increased trauma to the patient.

For surgical reconstruction, tissue may be moved from one part of the patient to another. These autografts (tissue transplants from patients) include skin grafts for burns, vascular grafts for cardiac bypass composition, and nerve grafts for face and hand reconstruction. Disadvantages of using autografts also include the need for combined surgery at the provider site and loss of function. In addition, surgical reconstruction often involves using body tissue for purposes not originally intended and can lead to long-term complications.

As a result of these shortcomings of existing treatment options, there is a need for engineered tissue equivalents, and it is the science of tissue engineering that has emerged as a new discipline. Its goal is to create tissue in culture not only for use as a model system in basic research, but more importantly, as an alternative tissue for damaged or diseased body parts. Of course, efforts to generate bioartificial tissues and organs for the treatment of humans date back to at least 30 years, and such efforts have approached clinical success only in the last decade. This is made possible by major advances in molecular and cell biology, cell culture technology, and material science.

The term "tissue engineering" has been relatively recent and used more widely in the past five years to describe the principles of engineering for the development of bioartificial tissues and organs and the field of alliances between different sectors applying life sciences.

One of the main strategies adopted for the generation of lab-growth tissues is the growth of isolated cells on a three-dimensional template or scaffold (matrix) under conditions that will treat the cells well and allow them to develop into functional tissues. When implanted, these bioartificial tissues must be structurally and functionally integrated into the body. The matrix can be formed from natural materials such as collagen or synthetic polymers such as plastics. Ultimately, the scaffold material must be biodegradable over time and act as an initial three-dimensional template for tissue growth.

As cells grow and differentiate in the scaffold, they will produce the various proteins needed to regenerate tissue. Degradation of the scaffold ensures that only natural tissue remains in the body. There are also other types of bioreactors that incorporate different techniques for making tissue from cells.

Virtually every tissue in the body is a potential target for biotechnology and the process takes place quickly at many fronts. For the skin as an organ, different types of engineering alternatives have been developed-the skin has been re-engineered using several different approaches with varying degrees of success.

U.S. Patent No. 5489304 describes non-cell transplantation with a synthetic outer layer bound to the collagen-chondroitin sulfate-derived skin like layer. Prior to the application of cultured epithelial autografts, this replacement, which is initially placed on the wound, has the drawback that it lacks growth factors or cells that can supply these factors important for skin wound healing.

U.S. Patent No. 5460939 describes another transplant that is a cell. Here, fibroblasts grow in the bio-resorbable lactic acid / glycolic acid copolymer mesh to form a sheet. In this transplant, the scaffolding mesh is not of natural origin.

Eaglstein & Falanga (1997) describe a skin graft comprising a skin layer with fibroblasts grown on bovine collagen matrix. In this transplant, the extracellular matrix is provided foreign to the cell, which produces human collagen, but the foreign component remains at the time of transplant application.

U.S. Patent No.5613982 describes a transplant in which human body skin is processed to remove antigenic cell components, leaving an immunologically inactive skin layer. It has the limitation that it is acellular and not readily available to human body skin.

In all of the above examples, the technical requirements for the production of equivalents are quite complex and will therefore increase the cost of the product. Cell sheets of fibroblasts using ascorbate have been developed, but the formation of such sheets requires about 35 days (Michel et al, 1999; L'Heureux et al, 1998).

Thus, materials for development are readily available, which are cells that do not have synthetic or natural foreign matrices that can cause an inflammatory response in some patients, and that allow them to produce growth factors and other proteins, which is relatively It can be produced in a shorter time, and its manufacture is technically simple so that there is a need for the development of a more cost-effective skin equivalent of the product.

An area that requires attention in the field of tissue-engineering products is bone replacement for patients who have fractures that are not treated and have left unbonded gaps. Autologous bone grafts increase trauma to the patient. Another approach is being attempted in bone engineering (Service, 2000). Biomaterials, such as collagen matrices infused with growth factors causing bone formation have been attempted, but such structures lack cellular components and the incorporation of the required amount of growth factors makes it a very expensive alternative. Ceramic or hydroxyapatite matrices seeded with mesenchymal stem cells are another approach, but the use of such scaffolds may not be ideal for the human body. Thus, there is a need for cost-effective cell implants that will result in bone healing.

Another area of concern in the field of tissue-engineering products is cartilage repair. It is known that articular cartilage has a limited capacity for complete recovery after injury. Cell-based therapies of autologous chondrocyte transplantation have shown good clinical results (McPherson & Tubo, 2000), but there is plenty of room for improvement since the time for complete recovery is very long. Possibly, preformed tissue rather than cell suspension will give better results for transplantation. In addition, preformed tissue is advantageous over free cells for surgical implantation. Thus, various approaches have been attempted to create cartilage-like structures using cells and scaffolds, but the ideal scaffolding matrix that will allow cells in the implant to mimic the natural cartilage formation process remains a challenge (Kim & Han, 2000). Thus, there is a need for the development of preformed tissue which can effectively lead to cartilage repair when implanted at the site of injury and which is also cost effective.

In summary, there is a need to develop relatively inexpensive living cell tissue substitutes for therapeutic purposes. The technically complex bioreactors mentioned earlier for developing three-dimensional tissue are expensive methodologies. Also, in general, there is always a need for the development of tissue substitutes, which, when tested, may prove to have better performance in one or more respects than existing substitutes.

Awaiting the need for time, the scientists of the present invention have developed a novel three-dimensional macroscopic tissue-like structure with the potential to be used as a tissue replacement in the human body. A novel property of these tissue-like structures is that no scaffold or foreign matrix is needed for tissue development, and the tissue can be formed entirely of cellular origin. In addition, other agents (except for high cell-seed-density) that aid in tissue formation, such as tissue-derived chemicals, tissue-induced growth factors, bottoms with special properties, rotational culture, are not adopted for tissue formation. There are no specific complex media requirements for forming tissue-like structures. A factor leading to the formation of macroscopic tissue-like structures is the large scale culture of cells at high cell seeding per unit area or space.

A crucial aspect of tissue engineering is how to assemble cells into tissues or three-dimensional structures. The present invention provides a novel method for achieving this.

4. Purpose of invention

In view of the above, it is therefore an object of the present invention to provide a novel method of collecting cells into three-dimensional tissue-like tissues and tissue-like structures.

In view of the above, it is therefore an object of the present invention to provide a three-dimensional macroscopic tissue-like structure for transplantation possible in the human body as a treatment for diseased or damaged conditions.

It is another object of the present invention to provide histologically effective macroscopic tissue-like constructs. "Histological competence" means that these tissue-like structures can be easily sectioned without cleavage.

Three-dimensional tissues of cells, such as engineering potential skin equivalents made of cutaneous fibroblasts, potential substitutes with fat stromal cell-induced osteogenic cells or bone-like properties made of osteoblasts, and potential substitutes for cartilage repair made from chondrocytes It is still another object of the present invention to provide a cost-effective provisional tissue equivalent made of fibrous cell cells of similar tissue and mesenchymal origin (at least). It is a related goal of the present invention to present the possibility of providing other tissues, which also has the property that these different cell types can undergo tissue-like substance formation in macromass culture as defined herein. If so, it is possible to be constructed from the corresponding cell type by the method of the invention.

It is still another goal of the present invention to provide three-dimensional tissue-like and macroscopic tissue-like structures of cells without the use of scaffolds or foreign matrices or complex bioreactors for tissue development.

Provides three-dimensional tissue-like tissues and macroscopic tissue-like structures of cells without the use of any agent that aids in tissue formation, such as tissue-derived chemicals, tissue-induced growth factors, floors with special properties, rotational culture, etc. It is still another object of the present invention.

Provide three-dimensional tissue-like tissue and other types of macroscopic tissue-like structures of cells, formed by using high cell seeding densities per unit area or space of culture vessel, without the requirement for any other agent to aid tissue formation. It is still another object of the present invention.

It is still another object of the present invention to provide macroscopic tissue-like structures with high cell density in the final form.

It is still another object of the present invention to provide tissue-like tissues and macroscopic tissue-like structures of cells that can be formed without the requirements of certain complex media components.

It is still another object of the present invention to provide tissue-like tissues and macroscopic tissue-like structures of cells in which their properties can be modulated to include desired properties by appropriate changes in growth and / or tissue-forming medium. Another purpose.

It is still another object to provide tissue-like tissues and macroscopic tissue-like structures of cells, which can be formed by macromass culture of other compatible growth surfaces according to requirements.

It is still another object of the present invention to provide a macroscopic tissue-like structure that can be scaled up to a simpler scale, i.e., to a larger size by macromass culture, in the two dimensions of the methods used for their formation.

Another object of the present invention is to provide three-dimensional tissue-like tissue at the microscopic level.

5. Description of invention

In the present invention, a method for collecting cells into three-dimensional tissue-like tissue by macromass culture and a novel method of macromass culture are provided. Provided are histologically competent macroscopic three-dimensional tissue-like structures generated by macromass culture of cells. The present invention relates to tissue engineering. More specifically, the present invention relates to the fabrication of three-dimensional tissue-like structures for transplantation possible in the human body as a treatment for diseased or damaged conditions. The present invention provides a method for the construction of cells into three-dimensional tissue-like forms and describes the tissue-like forms themselves.

Tissue fabrication is an important goal for the replacement of diseased tissue in the human body. Efforts are being made to explore and reinforce the tissue-forming capacity of cells for tissue engineering.

The process of tissue engineering of cell transplantation involves the following two main steps-

i. Obtaining the cells from an appropriate source. The cells obtained may require appropriate preparation such as differentiation into the desired cell type.

ii. Structuring tissue using appropriately prepared cells to make other tissue engineering products.

The present invention emphasizes the second of these steps. We have developed a simple and cost effective method for the generation of three-dimensional tissue-like tissues of cells and for the formation of live, cellular, potential tissue substitutes.

Tissue-like structures of the present invention are capable of withstanding physical manipulation and handling because they are necessary for the procedure of surgically positioning them in the body from the container holding them, with the aid of appropriate support and handling devices or instruments. Has a cohesive strength.

A substantial amount of work has been done to date in the generation of scaffold-based tissue replacements-these include scaffolds as important structural and often functional components. This scaffold is biocompatible, biodegradable (only natural tissue remains in the body) and is required to provide an acceptable environment for optimal cell function. From every possible point of view, the development of an ideal scaffold remains a challenge. Since the three-dimensional tissue-like structure generated by the present invention is made without the help of a scaffold, the present invention has the advantage that it avoids the need to integrate the scaffold. Formation of histologically competent tissue-like structures by the macromass method of the present invention does not require scaffolds. The tissue-like structures of the present invention therefore also eliminate any side effects or shortcomings that may be associated with the use of scaffolds that are not ideal in some respects. In the present invention, the extracellular matrix is synthesized by the cell itself and there is no foreign matrix component used. Tissue formation occurs by simply seeding cells at high cell density per unit area or space of culture vessel. It was termed a "macromass" culture, defined as a culture system for three-dimensional tissue-like formation or construction of cells, wherein the cells ranged from an area of about 10 ⁶ cells per cm ² to a unit area or There is no need for any other agent that is seeded at high density per space and aids in tissue formation. A broader definition of macromass cultures allows for the generation of macroscopic or microscopic, three-dimensional tissue-like tissues from cells by high-density cell seeding, bringing the cells together in certain advantageous ranges of high density of cells in three-dimensional space. Taken in close proximity, this is a way to promote the cohesive integration of cells into a three-dimensional tissue-like state, and there is no need for any other agent that aids in tissue formation. Certain high seeding densities of cells within an advantageous range are required to be achieved within a given space. In the macromass range of advantageous high cell seeding densities, when the cells are stabilized together in the three-dimensional space occupied by the cells at the bottom of the culture vessel, they are brought into close proximity to each other, causing or signaling them in the tissue formation mode. This results in a cohesive integration. (It can be noted that cell seeding densities in the macromass range could be achieved in vessels with flat or curved bottoms.) The results of using culture vessels with a diameter of about 0.75 cm or more for macromass culture resulted in macroscopic three-dimensional tissue. -Formation of a similar structure, where "macro" means that the size of the tissue is at least visually easy to identify with the human eye.

Previously known tissue culture systems, high-density micromass cultures have been used for cartilage differentiation of cells, and the range of such cultures has been limited to cell suspensions of 10-20 μl spots (Yoon et al, 2000). According to the practice, when cultured in vitro as a micromass culture, limb mesenchymal cells undergo the formation of total cartilage condensation or aggregates that exist as individual nodules covering the area of the micromass spot (Ahrens et al, 1977). The cell nodules thus formed are isolated from each other and microscopically with cells not formed in the middle. All cells are produced with the requirement of specific components added to the culture medium, such as growth factors, with larger, yet microscopic, mortar structures that together form one aggregate. However, the tissue-like materials produced by the inventors are macroscopic ( formed without the aid of any particular agent that aids in tissue formation ) and thus have a quality size that is required to have potential as a tissue replacement for the human body. In tissue-like tissues by the macromass culture of the present invention, all cells are thus part of a whole integrated tissue-like tissue; There is no individual nodule. By culturing, it was early found that leg prochondral mesenchymal cells made a nodular pattern (Downie & Newman, 1994). Wing wing cartilage mesenchymal cells made a sheet pattern by micromass culture; Unlike the macromass culture system of the present invention, it was in a serum free culture system. In addition, the limb prochondral mesenchymal cells could produce a sheet-like pattern, but again, unlike macromass culture, it was treated with TGFβ1 in serum-free medium, where any specific agent that aided in tissue formation was tissue-like sheet. It is not required for formation and there is no need for serum-free conditions.

To date, no question has been investigated whether cell-cell aggregation resulting in total tissue-like substance formation will be caused by high cell-seed-density culture, without any specific agent that aids tissue formation in the medium. However, our work has emphasized this problem and the present invention answers positively.

High-density cultures have been used to induce cartilage with microscopic individual nodule formation, but on a larger macroscale for the generation of macroscopic tissue for replacement in the human body, it has not been evaluated to date. Even on the microscopic scale, as mentioned above, in contrast to the macromass culture of the present invention, total aggregates are formed only with the help of certain agents.

In the first concept the term "macromass" may appear to mean a simple expansion of "micromass", but micromass has been developed as a method for the differentiation of cartilage of cells and also even a specific complex for microscopic whole mortar agglomerate formation. While containing media requirements, macromass is a method for the generation of three-dimensional tissue-like tissues and macroscopic tissue-like structures of cells, and in fact differs in the important sense that there are no specific complex media requirements for formation (below) As mentioned).

To date, efforts have been made towards the development of cell aggregates, the result of which is a microscopic material named mortar. Mortars are non-stick dishes (Takezawa et al, 1993), spinner-flask culture (Abu-Absi et al, 2002), polymer materials such as Eudragit (Yamada et al, 1998), Matrigel (Lang et al, 2001), Primaria dishes (Hamamoto et al, 1998), poly-D-lysine coated dishes (Hamamoto et al, 1998), proteoglycan coatings (Shinji et al, 1988), culture medium flow (Pollok et al, 1998), rotary culture ( Furukawa et al, 2001), liquid overlay technology (Davies et al, 2002), factors that enhance cell-cell adhesion such as insulin, dexamethasone & fibroblast growth factor (Furukawa et al, 2001), aggregation-promoting polymers- It is a three-dimensional cell structure made from hepatocytes and other cells with the help of various agents that aid in tissue-like formation, such as peptide conjugates (Baldwin & Saltzman, 2001), spin-wall bioreactors (Baldwin & Saltzman, 2001), and the like. Unlike these mortars, the tissue material made in our work is produced without the help of any such agent that aids in tissue-like formation. The above mentioned mortar is much smaller, mostly in the millimeter or sub-millimeter range. Since it is possible to make macroscopic tissue material by macromass culture as described in the present invention, they have a distinct advantage over mortar for positioning in the required position in the human body.

The largest of the mortars found in the published literature (about 1 mm in diameter) were formed by high-density pellet culture (Mackay et al, 1998). Their formation took place in the presence of serum-free media containing TGF-β3, dexamethasone, ascorbate 2-phosphate and insulin-transferrin-selenium supplementation, where such serum-free media was used for tissue development by macromass culture. Not required. In the preceding report using pellet culture of bone marrow induced mesenchymal helper cells, noculolytic formation did not occur in pellets cultured in DMEM + 10% FCS (Johnstone et al, 1998), whereas by macromass culture Tissue-like structures were found to form in DMEM + 10% FCS. Morphogenesis by micromass culture of multiple potential mesenchymal cells has been reported; Here again, aggregate formation occurred only when treatment of micromass culture with TGFβl or bovine bone extract (Denker et al, 1995). Microscopic bone cell morsels were reported to form in the presence of serum-free medium containing TGFβl (Kale et al, 2000; US Patent Application no. 20020127711) and culture of cells in the presence of TGFβl and in the presence of serum was reported as reported. In this method it is essential for bone cell morsel formation. In this operation, cell culture densities of about 1 × 10 ³ cells / cm ² to 1 × 10 ⁶ cells / cm ² have been described as advantageous for mortar formation. In addition to other features of the tissue-like structures of the present invention that distinguish it from the above operation, ie macroscopic, in addition to the absence of serum & no need of TGFβ1 for formation, advantageous seeding density ranges It is also different. In our work, tissue-like constructs did not occur at ¹ × 10 ⁵ cells / cm ² or less, whereas 1 × 10 ³ cells / cm ² of the work were 100-fold lower. Cell-agglomerates or nodules of osteogenic fetal stem cells were formed (Buttery et al, 2001), but again, as described herein, these nodules were microscopic and were not formed by high cell-seed-density culture. . The tissue material of the present invention that can be generated by macromass culture is macroscopic when performed on a large scale, and thus much larger in size than any mortar developed, without such specific complex media requirements for formation. Simple media such as DMEM + 10% FCS are sufficient for tissue-like construct formation by macromass culture. The cohesive plug of the cell is the chondrogenic cell US No. 5,723,331, which had been formed earlier, have preformed wells that have a surface that interferes with cell adhesion and thus does not allow cells of fixation, is a critical requirement for this, but in the present invention, however, such surfaces for the formation of tissue-like structures There is no requirement for this.

In one embodiment of the invention, the tissue-like sheet is formed from human skin fibroblasts as a potential skin substitute. Since it is known that human dermal fibroblasts are relatively non-immune when delivered to allogeneic recipients, dermal fibroblasts can be of allogeneic origin (US Pat. No. 5460939). This tissue-like sheet has the potential to be a skin equivalent, and the materials for it are readily available, which do not have a synthetic or natural foreign matrix that can cause an inflammatory response in some patients, which means that it is a protein that is different from growth factors. It is a cell that allows to produce and can be prepared after a relatively short time, the preparation of which is technically simple so that the product is more cost-effective.

In another embodiment of the invention, by macromass culture, a tissue-like material having bone-like properties is a potential tissue replacement that may have the potential to cause healing of small unbound gaps in bone fractures. From stromal cell-derived osteogenic cells. This may be a possible autologous treatment. This tissue-like material can have the potential to be a cost-effective cell implant, without the need for an extraneous scaffold, which can lead to the healing of small gaps in the bone. In the present invention, tissue-like constructs were also made of bone-derived osteoblasts.

In yet another embodiment of the present invention, tissue-like sheets have been developed from human chondrocytes as potential implants to induce cartilage repair in patients with articular cartilage damage. Since chondrocytes can be obtained from small biopsies of cartilage, autologous chondrocytes can be used for this tissue-therapy. This tissue-like sheet, when implanted at the site of injury, can effectively cause cartilage repair and also have the potential to be preformed tissue that will be cost-effective.

In a further embodiment of the invention, the microscopic three-dimensional tissue-like tissue is generated by macromass culture in a gelatin sponge.

In summary, the tissue-like constructs of the invention generated by macromass culture may have the potential to be living, cell, tissue substitutes without scaffolds and extracellular extracellular matrix, which are technically simple to make and thus It is cost-effective. Tissue-like constructs of the invention described in detail as tissue replacements for therapeutic purposes may also find other uses, such as in vitro drug testing and the like.

Preferred embodiments of the invention are further illustrated in the accompanying drawings, as described below.

(A) Dermal fibroblasts (b) Adipose stromal cells (c) Chondrocytes (d) Photographs showing macroscopic tissue-like structures formed by macromass culture in 3.5 cm dishes.

2. The cell-cell attachment process results in tissue-like structure formation that occurs in macromass cultures of adipose stromal cells (a) 1 hour after the start of macromass culture (b) 6 hours after the start of macromass culture.

Histological examination of tissue-sheets formed by macromass culture of skin fibroblasts (a) Hematoxylin & eosin staining shows three-dimensional tissue and (b) Masson-Trichome staining shows collagen synthesis.

4. Histological examination of tissue-like structures formed by macromass culture of osteoblast cells derived from adipose stromal cells (a) Hematoxylin & eosin staining shows three-dimensional tissue and (b) Masson-Trichome staining .

5. Histological examination of tissue-like structures formed by macromass culture of chondrocytes (a) Hematoxylin & eosin staining shows three-dimensional tissue and (b) Masson-Trichome staining.

6. Collagen type I immunostaining of histological sections of tissue-like structures made from dermal fibroblasts, showing positive detection of collagen type I.

7. Cells regrowed from isolated tissue sheets made from skin fibroblasts by macromass culture to assess viability.

8. Gene expression analysis of tissue sheets formed from skin fibroblasts by macromass culture, assayed by Reverse Transcriptase-PCR. RT-PCR products corresponding to various genes known to be important for the skin's wound healing process appear to be electrophoresed on 2% agarose gel (M) DNA molecular size markers (1) collagen type I (2) syndecan 2 ( 3) tenasin-C (4) vascular endothelial growth factor (5) collagen type III (6) fibrinogen (7) keratinocyte growth factor (8) transforming growth factor 1β.

9. Gene expression in tissue-like constructs made from osteogenic cells derived from adipocytes. RT-PCR products show electrophoresis on 2% agarose gel (M) DNA molecular size markers (1) collagen type I (2) osteopontin (3) parathyroid hormone receptor (4) bone morphogenetic protein 2 (5) Bone Morphogenic Protein 4 (6) Bone Morphogenic Protein Receptor IA (7) Bone Morphogenic Protein Receptor IB.

10. Gene expression in tissue-like constructs made from chondrocytes. RT-PCR products are electrophoresed on 2% agarose gel (M) DNA molecular size markers (1) aggrecan (2) collagen type II (3) collagen type X.

Figure 11. Derived from fat stromal cells in the presence of regulated bone formation medium showing (a) local actual bone formation (Masson-Trichome) and (b) local calcium deposition (Von Kossa) in tissue-like structures. Histological analysis of tissue-like structures made of osteogenic cells, demonstrating that the properties of tissue-like structures made by macromass culture can be controlled by changes in the medium.

Figure 12. (a) DMEM + 10% FCS (b) Histological sections of tissue-like structures made from chondrocytes in the presence of cartilage medium (Toluidine Blue staining), (b) Cartilage-specific extracellular matrix formation It is shown that the properties of tissue-like structures made by macromass culture can be controlled by changes in the medium.

13. Histological sections of complex subjects (Masson-Trichome staining), consisting of tissue-like sheets made from skin fibroblasts and collagen + fibrous gels, tissue-like tissues made by macromass culture may be a component of the subject Prove that.

Figure 14. Histological sections of macromass cultures (hematoxylin & eosin staining) in gelatin sponges show colonies of microscopic three-dimensional tissue-like tissue formed.

Various other aspects of the invention are described in more detail in the following sections.

7. Materials and Methods:

By the inventor of the present invention, throughout the description of the invention and the appended claims, although the area of the culture vessel is mentioned in terms of the diameter of the circular culture vessel, the aspect actually includes any type of culture vessel. It is clearly described that its area is the same as that of the circular container of the mentioned diameter. In addition, although the operations presented herein relate to containers with flat bottoms, macromass cultivation could be accomplished in culture containers with flat or non-flat bottoms.

7.1. Cell Separation, Media and Culture-

In the present invention, human skin fibroblasts were isolated from human skin biopsy. The dermis was separated from the epidermis by treatment with dipase (Sigma, St. Louis, USA). The dermis was finely chopped and digested overnight with 0.01% collagenase in DMEM + 10% FCS and the cells were then attached. Cells were incubated in DMEM + 10% FCS in 5% CO ₂ at 37 ° C. and subcultured with trypsin-EDTA solution. Adipose stromal cells were isolated from human liposuction material according to the protocol described by Zuk et al. (2001). These cells were maintained in DMEM + 10% FCS. These cells were induced to osteogenic differentiation according to the protocol described by Zuk et al (2001). Prior to incubation in maintenance medium of DMEM + 10% FCS, chondrocytes were isolated from human cartilage pieces by crushing the cartilage and treating with collagenase. Osteoblasts were isolated from human bone by a similar procedure and maintained in DMEM + 10% FCS with 50 μg / ml ascorbic acid. Regulated bone formation medium was prepared by using a medium in which the osteogenic fat stromal cells were growing as part of the bone formation medium for the same cells in the next secondary culture. Cartilage media were prepared according to Zuk et al (2001).

7.2. Form tissue-like structures-

Formation of tissue-like constructs was achieved by macromass culture, which is a novel method of the present invention as defined earlier in the description. Cultured cells were harvested using trypsin-EDTA. They are resuspended in an appropriate volume of medium and are seeded in a culture dish having a well diameter of 3.5 cm, preferably at a seeding density of about 10 cells per cm ² or at a tissue-forming cell density in the macromass advantageous range. Thus, preferably about 10 ⁷ cells were seeded in a single well of 6 well plates (9.6 cm ² area) for formation of a single tissue. For smaller or larger tissue-like structures, the total number of seeded cells was adjusted to achieve seeding densities favoring tissue-like tissues.

7.3. Histological Analysis-

Upon formation, tissue-like structures were fixed and processed for histological examination. Von Kossa staining and Alcian blue staining were performed on the appropriate tissue constructs of the present invention, according to the methods described by Zuk et al (2001) and Bancroft et al (1994). Oil Red O staining was performed by the method described by Bancroft et al (1994). Toluidine Blue staining was performed as follows-after deparaffinization and hydration of the sections, they were stained with 2% aqueous Toluidine Blue solution for 1 minute and then washed in water for 2-3 minutes. Sections were then dehydrated at 2 changes in 100% acetone, then cleared and mounted in xylene.

Other histological processes were performed by Histopathology Laboratory at Sir Hurkisondas Nurrotamdas Hospital & Research Centre, Mumbai, India.

7.4. Immunohistochemistry-

Collagen type I immunostaining was performed on sections of paraffin-infiltrated tissue using goat anti collagen type I antibodies and ABC staining system of Santa Cruz Biotechnology, Santa Cruz, USA.

7.5. Vitality of cells in formed tissue-like structures-

The tissue material of the present invention was chopped, digested with 0.5 mg / ml collagenase in serum-free DMEM for 15 minutes, and the released cells were resuspended in growth medium. Alicottes were stained with Trypan Blue. Cells were seeded in culture flasks to assess viability.

7.6. Gene Expression Analysis-

Gene expression in tissue-like constructs made from skin fibroblasts, osteogenic adipocytes and chondrocytes was analyzed by Reverse Transcriptase-PCR. RNA was extracted from tissue-like structures using Trizol (Gibco-BRL, Grand Island, USA). RT-PCR was performed using primers specific for each gene and the Titan One-Tube RT-PCR system (Roche, Mannheim, Germany).

7.7 Collagen + Fibrin Gel Preparation-

For the preparation of collagen + fibrin gel, 134 μl 3.33 mg / ml rat tail collagen type I in 0.1 ISI acetic acid (Sigma, St. Louis, USA), 8 μl 4N NaOH, 165 μl 28.8 mg / ml fibrinogen in 1 × DMEM , And 210 μl of 1.48 mg / ml thrombin in 1.66 × DMEM. The mixture was gelled at 37 ° C. for 2 hours.

7.8 Tissue Formation by Macromass Culture in Gelatin Sponges

The gelatin sponge used was AbGeI (Sri Gopal Krishna Labs. Pvt. Ltd, Mumbai, India).

8. Results and Discussion:

8.1. Formation of three-dimensional tissue-like and macroscopic tissue-like structures

By macromass culture, tissue-like material (FIG. 1) from skin fibroblasts in the presence of DMEM + 10% FCS was formed in the shape of a sheet detached from the growth surface by gently peeling off spontaneously or with a blunt instrument. Adipose stromal cells also formed tissue-like material in DMEM + 10% FCS that was negative for lipids by Oil Red O staining. Because this is the case, in order to make possible useful tissues from adipose stromal cells, they have differentiated into osteogenic cells, which have formed a similar sheet in macromass cultures, which will shrink into tight material when removed and further cultured. Can be. Chondrocytes isolated from human cartilage also formed such tissue sheets upon macromass culture in DMEM + 10% FCS. After rinsing the sustained medium containing ascorbic acid, osteoblasts isolated from human bone also formed tissue-like structures by macromass culture in DMEM + 10% FCS. As seen from the extensive formation of extensions from cell and cell integration, shown in FIG. 2, cell integration appears to play an important role in the formation of such tissue-like structures. Much larger sheets were formed when the scale of the dermal islet hair cell macromass culture was expanded by seeding the cells at the seeding densities mentioned in the 8.5 cm Petri dish. Thus, it appears that macromass culture can be directly scaled up in the area direction to obtain tissue as large as desired. Dermal fibroblasts also formed sheets by macromass culture in serum-free DMEM, but the time of formation was greater than in DMEM containing 10% FCS when compared to in serum-containing medium overnight at 3-4 hours. In serum-containing medium, the time of formation of tissue from dermal fibroblasts, from adipocytes & osteogenic cells derived therefrom was about 4 hours, and about 18 hours from chondrocytes. After washing the cells to form osteogenic medium containing ascorbic acid, osteogenic cells derived from adipose stromal cells formed tissue-like material in bone formation medium as well as in DMEM + 10% FCS. The generation of tissue-like structures by macromass culture was successfully performed in culture vessels with a diameter of 0.75 cm to 8.5 cm. Macromass culture in culture vessels smaller than 0.75 cm diameter and larger than 8.5 cm diameter may be extrapolated to result in the formation of smaller or larger tissue-like structures, respectively. Thus, the dimensions of the three-dimensional tissue-like structure can vary.

Although these tissue-like structures are not fully formed tissues, they are capable of restoring and regenerating stem cells or transplants of partially differentiated cells (which are not fully formed tissues but at the very beginning of tissue formation) (Kaji & Leiden, 2001). In a similar way to the discovery that it could lead to the body's healing process.

It was found that the phenomenon of three-dimensional tissue-like material formation by macromass culture was dependent on cell seeding density. To investigate whether tissue formation all occurred at high densities, dermal fibroblasts were seeded at a range of different cell densities per unit area. Type of sheet formation ² cm It was found that tissue sheets did not form at seeding densities of up to 6.66 × 10 ⁴ (less than 5 times) cells per cm ² , while occurring at about 3.33 × 10 ⁵ cells per sugar. In addition, cm ² per 3 x 10 ⁶ natjiman the tissue sheet formation occurred in the seeding density of cells per cm ² 7 x 10 ⁶ cells than did not take place; At the latter seeding density the cells only loosely aggregated together but did not form a coherent tissue material. Thus, the tissue sheet formation is 3.33 x 10 ⁵ cells per cm ² and 3 x 10 ⁶ cells per cm ^2, as well as all took place in the density between the two values tested. At densities tested above or below this range, no tissue formation occurred. Similar experiments were performed with natural adipose stromal cells and chondrocytes and the results are shown in Table 1. As in dermal fibroblasts, there was a minimum and maximum tissue-like construct formation seeding density for these cell types and was also similar to that of dermal fibroblasts. These data indicate that the minimum and maximum cell seeding density per unit area is present for tissue formation by macromass culture. The range of high cell densities in which tissue formation is caused by macromass culture may differ for other cell types not tested.

The lower seeding densities of the cells used as shown in Table 1 resulted in thinner, ie, tissue-like structures with smaller three dimensions, while using higher seeding densities resulted in thicker, i. Resulting in tissue-like structures. Thus, the dimensions of the three-dimensional tissue-like structure can vary.

8.2. Histology (Figures 3,4,5)-

Hematoxylin & eosin staining of sections of other tissue-like structures of the present invention shows three-dimensional structural construction and extracellular matrix formation. Masson-Trichome staining of tissue-like structures formed from skin stromal cells as well as natural stromal cells and osteogenic stromal cells showed collagen synthesis. Collagen type I immunostaining of tissue-like structures made from skin fibroblasts, cultured for 10 days, showed positive staining for collagen type I (FIG. 6). Thus, extracellular matrix formation appears to occur in tissue-like structure formation by the method of macromass culture.

8.3. Vitality of cells in formed tissue-like structures

Cells re-isolated from tissues formed from skin fibroblasts and chondrocytes had at least 98% viability by Trypan Blue staining; Cells from tissue material from adipose stromal cells were about 90% viable. Regrowth was assessed by plating the isolated cells and clots and found to be viable in each case, as shown in FIG. 7 showing cells growing out of the clots of dissociated skin fibroblast tissue sheets.

8.4. Expression analysis of tissue-like structure-

To assess whether tissue sheets formed from skin fibroblasts have potential as dermal replacements, the expression of genes known to play an important role in the wound healing process of the skin was analyzed (FIG. 8). Collagen type I, collagen type III, keratinocyte growth factor, TGFβ1, fibrinocytes, vascular endothelial growth factor, tenascin-C and syndecane-2 have been shown to be expressed in tissue sheets and thus made from dermal fibroblasts This tissue-like sheet demonstrates its potential as a dermal replacement. To assess whether tissue-like structures made from osteogenic fat stromal cells have potential as bone-like tissue substitutes, expression of bone-specific expressed genes was analyzed. Tissue-like structures have been shown to express collagen type I, osteopontin, parathyroid hormone receptors, bone morphogenic protein 2, bone morphogenic protein 4, bone morphogenic protein receptors IA, IB & II, and thus In addition to the localized calcification mentioned and the actual bone invasion formation, it demonstrates its bone-like properties (FIG. 9). Similarly, tissue-like structures made from chondrocytes have been evaluated for their potential as cartilage tissue substitutes. Cartilage-specific expressing genes Collagen Type II, Aggrecan and Collagen Type X have been found to be expressed and therefore, as mentioned below, in addition to the formation of a cartilage-specific extracellular matrix, its cartilage-like properties. Prove (Fig. 10).

8.5. Modulation of the properties of tissue-like structures by the flexibility of tissue-forming medium

In macromass culture, as long as tested, tissue formation is independent of the medium conditions, as long as nothing inhibits the tissue-like tissue by the macromass, since tissue formation is independent of the medium conditions, it may be possible to It is possible to tailor the nature of the organization. This results from tissue sheet formation by dermal fibroblasts in both serum-containing and serum-free media, as well as tissue formation from fat stromal cells in both DMEM + FCS and bone formation medium containing dexamethasone and β-glycerophosphate. It is obvious. Thus, the properties of the formed tissue can be adjusted to incorporate the desired properties by altering the tissue-forming medium and / or growth medium of the cell. For example, as presented herein, bone-like properties in the form of actual bone formation were induced in tissues formed from adipose stromal cells by culturing the cells and forming tissue-like structures in a controlled bone formation medium. In comparison with the bone formation medium alone, no actual bone osteotomy was seen (FIG. 11). Von Kossa staining of tissue material formed from osteogenic stromal cells in the presence of regulated osteogenic medium showed localized areas of calcification, thus demonstrating bone-like properties, while osteogenic substrate in non-regulated osteogenic medium Constructs made from cells did not exhibit these properties. Another example of such adjustment is cartilage medium in the presence of DMEM + 10% FCS or containing 1% FCS and insulin; And the formation of tissue-like structures from chondrocytes in the presence of TGFβ1 as a cartilage inducing agent. Properties with extracellular matrix properties of the cartilage phenotype occurred in tissue-like structures made in cartilage media, but those made in DMEM + 10% FCS did not have such properties. This is shown by Toluidine blue staining in FIG. 12. Thus, since tissue formation by macromass culture remains without specific complex media requirements, such specific components can be used in media for controlling the properties of the tissues in which they are formed. It also follows from the above results that tissue-like tissues and macroscopic tissue-like structures can be formed in the presence of other culture media, wherein the other media presented here are examples and the present invention is not limited to these examples in this regard.

8.6. Growth Surfaces for Macromass Culture-

This is undesirable because tissue-like sheets formed from cells plated on the plastic surface tend to spontaneously fall and curl or curl, and the sheets do not straighten once rounded. Tissues developed as possible dermal replacements are required to remain upright. To this end, macromass incubation of skin fibroblasts was performed on a Hybond-N (Amersham Pharmacia Biotech, Buckinghamshire, UK) filter mounted on a plastic dish. In this adaptive form, the sheet formed to remain upright and attached to the filter can thus be applied to the skin wound later on, with the filter upside down. This experiment demonstrates that it is possible to achieve tissue formation on other compatible growth surfaces by macromass culture. Thus, in this case, the sheet made by the macromass culture becomes a component of the potential implant, including a nylon filter that serves as a support layer and its requirement is not as a scaffold for the tissue-like tissue of the cell. . This support layer is not designed to be integrated into the body with the dermal-like sheet, so it is not a scaffold to be part of the body, but a support handling device for the application of the dermal-like sheet. This support layer will be removed after healing.

8.7. Tissue-Like Structures as Components of Subject—To demonstrate that tissue-like structures can be integrated (and larger) into components or parts of a subject, skin fibroblasts are seeded by macromass culture to produce tissue-like sheets. Formed, and then after the culture medium was removed, it was covered with collagen and fiber gel mix. The gel then hardened. Now, the gel could be lifted with a tissue-like sheet attached to one side of it. The histological section of this complex, in which the tissue-like structure is a component, is shown in FIG. 13. Apart from gels and sheets, other matrices, such as sponges or membranes, could also support tissue-like-structures from one side by attaching them. Thus, tissue-like tissues and macroscopic tissue-like structures can be combined with other objects, such as sheets or membranes, gels, sponges, or other matrices.

8.8. Microscopic three-dimensional tissue-like tissue- by macromass culture

Skin fibroblasts were seeded onto a 3.5 cm diameter gelatin sponge at DMEM + 10% FCS at a seeding density of about 2.5 × 10 ⁶ cells per cm ² of sponge area. For gelatin sponges, this application of the high cell-seed-density macromass culture method results in the formation of microscopic clusters of three-dimensional tissue-like tissue in the sponge, as shown in the histological section of the sponge shown in FIG. 14. It was. Thus, the method of macromass culture can be used to generate three-dimensional tissue-like tissues of cells at the microscopic level, and tissue-like tissues are also components of the entire aggregate.

9. Conclusion:

The novelty of the present invention is high cell-seed, as well as other important features, such as the fact that no scaffolding material is employed or no specific agent is required to aid in tissue formation / complexing medium preparation, as detailed above. Density cultures can produce three-dimensional tissue-like tissues of whole cells without any specific agent that aids tissue formation and can induce such tissues and increase in area direction to produce other types of macroscopic three-dimensional tissue-like structures It exists in the fact that it can. The inventors first report that whole three-dimensional tissue-like tissues of cells and other types of macroscopic three-dimensional tissue-like structures can be produced by high cell-seed-density culture alone.

It may be possible for other mesoderm cells or cells of endoderm or ectoderm origin to also form such tissue-like tissue by macromass culture. Thus, tissue-like tissues and macroscopic tissue-like structures made by macromass cultures from certain types of mammalian cells, if they are possible, are within the scope of the present invention and the defining features are limited to methods of macromass culture. Tissue-like organization achieved by.

Although macroscopic tissue-like constructs are a preferred embodiment of the invention, macromass cultures on smaller scales, ie, anything smaller than about 0.75 cm diameter in the culture area will result in smaller tissue-like tissues, and macromass cultures It is clear that when the size of is reduced, the size will decrease toward becoming microscopic.

Microscopic tissue-like tissues by high cell-seed-density macromass culture, either in this form or in other forms such as the gelatin sponges described earlier, are therefore also within the scope of the present invention. Likewise, tissue-like structures can be generated by macromass culture in culture vessels larger than 8.5 cm in diameter.

Thus, in the foregoing description, certain embodiments of the present invention have been described; Examples include the use of different cell types, the use of alternative growth surfaces, the use of changes in the medium to adjust the properties of the formed tissue, the increasing rate of tissue formation, the size of the culture vessel, the range of tissue-forming high cell densities, and A perspective of the production of potential implants in which the tissue made by macromass cultivation has been presented. Although only the described embodiments have been presented, they serve only for the purpose of example or illustration and do not limit the invention.

It is to be understood that other variations or substitutions may be made to the invention, which will be within the scope of the invention. For example, we believe that one such modification is the capture or encapsulation of a tissue material made by macromass culture into a suitable gel or matrix, or another such modification may be achieved by any means in which multiple tissue-like materials or sheets are brought together. Plan to be a structure that is joined or maintained by In such constructs, tissue-like constructs made by macromass culture will now be a component of the total replacement. This is in a different manner than those shown in the results section, whereby tissue-like tissues and structures generated by macromass culture can be components of a subject. As evidenced in the results, scaffolds are an example because tissue-like tissues of cells by macromass culture do not require any scaffolds, and histologically competent tissue-like structures are formed without the help of scaffolds. For example, it can be provided for macromass culture as an alternative growth surface such that the scaffold is part of the total replacement. Thus, the three-dimensional tissue-like tissues and histologically competent tissue-like structures of the present invention can be developed without scaffolds, but with appropriate scaffolds provided they provide advantageous properties or advantages over tissue-like structures alone. Can be combined together. Other modifications include the use of other cell types that have the property of forming tissue-like material by macromass culture, the use of other compatible growth surfaces than those described, other media changes for adjustment, and different magnitudes of increase in rate, etc. Will be Accordingly, the description is not intended to limit the invention to the precise illustrative embodiments disclosed.

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Claims (24)

Without the need for any other agents that aid in tissue formation, cells are seeded at a high density per unit area of the culture vessel, over a range of about 10 ⁶ cells per cm ² , resulting in three-dimensional tissue-like formation or composition of the cells. A culture system, Providing tissue-like structures made of mesodermal cells and applicable to other cell types, A method of producing living tissue-like tissue of cells, ie, a method of culturing macromass comprising three-dimensional tissue-like structures, without the need for scaffolds or foreign matrices. The method of claim 1, further comprising: generating macroscopic or microscopic, three-dimensional tissue-like tissue from the cells by high-density cell seeding and Bringing the cells together in a certain advantageous range of high density of cells in a three-dimensional space to facilitate cohesive integration of the cells into a three-dimensional tissue-like state, without the need for any other agents that aid in tissue formation. Included, A method comprising using a macromass culture comprising a culture system for tissue formation. The method of claim 1, comprising forming a tissue-like sheet from cells comprising human fibroblast cells of mesenchymal origin, wherein the tissue-like sheet is a synthetic or natural outpatient capable of causing an inflammatory response in some patients. Is a cell which is free of matrices and which allows it to produce growth factors and other proteins, which can be produced in a relatively short time. Engineering potential skin equivalents made from cutaneous fibroblasts, potential substitutes with bone-like properties made from osteogenic cells derived from adipose stromal cells, and potential cartilage substitutes made from chondrocytes, but not limited to these cell types Not; Generating a tissue-like tissue of cells that can be made to take other forms, Tissue of cells comprising the macroscopic three-dimensional structure according to claim 1 for use as a tissue substitute for transplantation, wound healing, as an in vitro model for drug testing, etc., made from fibroblast cells of mesenchymal origin -Like organization. 5. The tissue-like tissue of claim 4, wherein the cell comprises a three-dimensional and three-dimensional macroscopic tissue-like structure in nature. The method according to claim 4, which can be made to take other forms, which are created for the purpose of achieving different properties or properties, said other forms: 3D macroscopic tissue-like structures themselves, wherein the term "macro" means that the size of the tissue is at least visually easy to identify with the human eye, and the macroscopic tissue-like structures Is histologically competent; Combining the three-dimensional tissue-like tissue with another matrix, such as a gel, sheet, membrane, or sponge, or with another scaffold or the like; The tissue-like tissue of a cell, characterized in that the form of microscopic three-dimensional structure. 5. The tissue-like tissue of a cell according to claim 4, comprising a tissue-like tissue which can be generated solely using the cell alone and the culture medium. The method of claim 4, wherein the tissue-like tissue of the cell is Tissue-induced chemicals such as ascorbic acid; Tissue-induced growth factor; And Floor with special properties; Rotational culture; Complex bioreactor; Or foreign scaffold or matrix or support Tissue-like tissue of a cell, characterized in that it can be produced without using any agent that aids tissue-forming. 5. The tissue-like tissue of a cell according to claim 4, comprising a tissue-like tissue that can be produced without the need for an extracellular matrix component. The method of claim 4, wherein Skin fibroblasts; Fat stromal cells; Osteogenic cells derived from adipose stromal cells; Chondrocytes; And osteoblasts Tissue-like tissue of cells, characterized in that it is produced from different cell types. 5. The macromass culture of claim 4, wherein there is a range of cell seeding densities that promote tissue formation for its formation and the exact range of tissue-forming high cell seeding densities may vary for different cell types. Tissue-like tissue of cells produced by. 5. The tissue-like tissue of a cell according to claim 4, wherein the time of formation can vary according to different cell types or different media conditions. The method of claim 4, wherein Serum-free medium conditions; And Comprising a specific composite medium composition, Tissue-like tissue of cells produced by macromass culture, characterized in that it is produced without the need for specific medium conditions. The method of claim 4, which is three dimensional Different three-dimensional size; Larger or smaller tissue-like structures by increasing or decreasing the rate of macromass culture; And Flexibility in dimensions including variable sizes or scales by varying the number of cells used to achieve seeding density within the macromass advantageous range of tissue-forming density by increasing or decreasing the rate of macromass culture. Tissue-like tissues and other forms thereof of cells produced by macromass culture. The method of claim 4, wherein Formation of tissue-like tissue in the presence of other culture medium, other growth and / or tissue-forming medium; And Provided that the addition of ingredients does not negatively affect tissue formation, the inclusion of these ingredients in the growth and / or tissue-forming medium, or, if such changes in medium do not adversely affect tissue formation, by changing the medium. Controlling the properties of the tissue-like tissue, Tissue-like tissues of cells and other forms thereof characterized by having flexibility with respect to the culture medium used. 5. The tissue-like tissue of claim 4, comprising a tissue replacement achieved at another compatible growth surface or scaffold. 5. The tissue-like tissue of claim 4, comprising the manufacture of tissue-like structures made in any type of culture vessel, with flat or curved bottoms. The method of claim 4, wherein Can be made to take different forms, different forms being created for the purpose of achieving different properties or characteristics, Three-dimensional macroscopic tissue-like structures with dimensions that are easily visually identifiable to the human eye, Histologically capable macroscopic tissue-like structures; Combining the three-dimensional tissue-like tissue with another matrix, such as a gel, sheet, membrane, or sponge, or with another scaffold; Tissue-like tissue of a cell, characterized in that the form of microscopic three-dimensional structure. Employing high cell-seed-density cultures to produce tissue-like tissues of cells without the need for the adoption of specific agents and scaffolds to aid tissue formation; Providing a tissue-like construct made from mesodermal cells, but not necessarily limited to these cell types; Creating a tissue-like tissue of the cell and structuring the tissue-like tissue of the cell to form another tissue engineering preparation by forming a live, tentative tissue substitute, A method for the generation of tissue-like tissues of cells comprising the fabrication of three-dimensional tissue-like structures without the help of a scaffold. 20. The method of claim 19, comprising using a high cell seeding density per unit area or space of the culture vessel without the need for other agents to form tissue-like tissues of cells and provide macroscopic tissue-like structures. . 20. The method of claim 19, comprising forming tissue-like tissue using macromass culture by seeding cells at high cell density per unit area or space of culture vessel. The method of claim 21, wherein the macromass culture is culture for tissue formation, including seeding the cells at a high density per unit area or space of the culture vessel, over a range of about 10 ⁶ cells per cm ^2, and without the need for other agents to assist in tissue formation System, The macromass culture is further: By high-density cell seeding, generating macroscopic or microscopic, tissue-like tissues from the cells, without the need for any other agents that aid in tissue formation, certain advantageous ranges of high density of cells in three-dimensional space Taking together very closely together; The high cell seeding density is advantageous because the cells are stabilized together in a three-dimensional space occupied by the cells at the bottom of the culture vessel so that they are in close proximity to each other, causing them to signal or signal them in tissue formation mode. Achieving a macromass range; And Achieving a macromass range of cell seeding densities in a container with a flat or curved bottom and thereby using a culture vessel of about 0.75 cm in diameter for macromass culture results in the formation of macroscopic tissue-like structures Means defining a size of tissue that is easily visually identifiable to the human eye. Made according to methods for generating macroscopic tissue-like structures and whole tissue-like tissues of cells without the need for any specific agent to induce tissues, and of high cell-seed-density culture, ie of mesenchymal origin Implanting into a human or mammalian body, by macromass culture from cells containing them, can be increased by a constant rate without the need for scaffolding materials and specific agent / complex media formulations to produce macroscopic tissue-like structures As a tissue-like structure for Tissue-like tissues and tentative tissue equivalents of the resulting cells are engineered temporary skin equivalents made from skin fibroblasts, potential substitutes with bone-like properties made from adipose stromal cell-derived osteogenic cells or osteoblasts and cartilage made from chondrocytes. Tissue-like structure, characterized in that it is made from cells of mesenchymal origin, including a potential substitute for recovery. The method of claim 23, wherein Tissue-like tissues and macroscopic tissue-like structures of cells are scaffolds for tissue generation, for the production of three-dimensional tissue-like structures for implantation into human or mammalian bodies as a treatment for diseased or damaged conditions or Made without the need for an exotic matrix or complex bioreactor; Formation of tissue-like structures is free of the need for preformed wells with surfaces detrimental to cell attachment; Tissue-like tissues and macroscopic tissue-like structures of cells are characterized in that tissue-induced chemicals, tissue-induced growth factors, floors with special properties, are made without the need for any agent, such as rotational culture. Similar structures.

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