TWI622649B - Preparation of parental cell bank from foetal tissue - Google Patents

Abstract

The invention relates to a method for preparing a parental cell bank (PCB) from fetal tissue in vitro. The fetal tissue is composed of fetal epiphysis tissue, fetal Achilles tendon tissue, and fetal skin tissue. The method uses a fast, physical primary Cell culture selection method to select cell types to be used in wound and tissue repair methods.

Description

Preparation of a parental cell bank from fetal tissue

The invention relates to a method for preparing a parental cell bank (PCB) from fetal tissue in vitro. The fetal tissue is composed of fetal epiphysis tissue, fetal Achilles tendon tissue, and fetal skin tissue. The method uses fast and physical primary cells. Culture selection method to select cell types to be used in wound and tissue repair methods.

Cell therapy has become an additional option of interest as a medical therapy for repairing, restoring, or improving tissue function, and in particular as a medical therapy for repairing, restoring, or improving tissue function in combination with conventional surgical techniques. For patients, some cell options are more suitable for cell therapy than others. For animal and human tissue selection at all developmental ages, their advantages and disadvantages can be assessed for each final cell type. Currently, the limitations on therapies for human cell lines are technical limits regarding: cell proliferation capacity (simple culture conditions), maintaining differentiated phenotypes of primary human cell cultures, transmission of infectious diseases, and selected cell populations Depends on the consistency and stability of the separation procedure. Cultured primary fetal cells from a single organ donation fit the urgent and rigorous technical point of view for the development of medical products. A master cell bank and a working cell bank from a single fetal organ donation can be developed in a short period of time, and safety tests can be implemented at each stage of the cell bank production.

For surgical procedures and tissue engineering of specific tissues, cell therapy has been proposed as a less invasive alternative therapy or as a combination therapy. Some cell types have been investigated for use in cell therapy: embryonic stem cells (ES), umbilical cord cells, fetal cells and adult stem cells (from bone marrow-hematopoietic stem cells or HSC and bone marrow stem cells or MSCs), as well as adipose tissue, platelets, Placenta and amniotic fluid cells. For all applications in tissue engineering, the source and type of cells is the main focus. For specific therapies with various advantages and disadvantages, different types of cells need to be processed in different ways to manipulate their ability to differentiate and self-renew.

Fetal cells have been widely used in biology and medicine for many years, but there is not much known about their importance, especially in developing the necessary vaccines. Fetal cells are differentiated cells that are highly expandable, regenerative, and low immunogenic. It can be isolated from fetal tissues after 9 weeks of embryonic development. Fetal skin cells provide the ideal solution for effective and safe cell therapy and tissue engineering, including the following reasons: a) the ability to expand cells from a single organ donor; b) the need for cell growth is minimal; c ) Adapted to the biological material being delivered; and d) resistant to oxidative stress. Fetal skin cells are powerfully expandable because they only require a single organ donation (1-4 cm ² tissue) to create enough frozen cells to produce a cell bank that can be used for hundreds of thousands of treatments (i.e. , For the skin, more than 35 billion 9 x 12 cm fetal skin structures can be generated from a single dedicated cell bank). Moreover, compared to stem or mesenchymal cell types, the requirements for cell culture are minimal. Because fetal skin cells have been differentiated and do not need to be directed or changed, many of the additional growth factors that are normally required are not required for cell culture and expansion. For the production of cell banks, careful selection of donors and extensive screening of donors and cultured cells to avoid spreading viral, fungal or bacterial diseases, fetal cells can be used safely and without risk for medical purposes. In addition, fetal cells, unlike newborn cells, young cells, or adult cells, are particularly adapted to biological materials and can therefore be efficiently and easily delivered to patients. It has been reported that cells from donors (newborn to adult) cannot be efficiently integrated into various biological materials, and some biological materials are actually toxic to cells. Although scaffolds are really important for tissue engineering, the cell type is most likely the limiting factor. In order to handle end products for clinical delivery, homogeneous distribution and rapid development of end products are major significant advantages. If a long incubation period is required, there will be a non-negligible increase in the risk of contamination for autologous transplantation or hitherto commercially available products. It is also important to have a consistent and easily repeatable process. By developing a consistent cell bank with fetal cells, many risk factors can be eliminated, and clinically safe and effective treatments for human cell lines can be provided.

For cell line products, the factors including identity, purity, sterility, stability, stability and effectiveness are recommended. In summary, the new regulations have strict guidelines for manufacturing and the environment used to manufacture cell line products used in clinical trials and treatments. Current limitations on therapies for human cell lines are in cell proliferation capacity (simple culture conditions), maintenance of differentiated phenotypes in primary human cell cultures, transmission of transmissible diseases, and selected cell populations as separate Technical constraints in terms of consistency and stability of steps. Cultured primary fetal cells from a single organ donation An eager and rigorous technical perspective for developing medical products. From a single fetal organ donation, a seed cell bank and a working cell bank can be cultured in a short time, and safety tests can be performed at all stages of cell bank production. For medical purposes, fetal cells can use up to two-thirds of their lifespan in scale-up processing, and can ensure consistency in several biological properties including protein concentration, gene expression, and biological activity.

Fetal cells are relatively simple to collect, expand and store, making fetal cells an attractive option for cell therapy. Unlike ES cells, fetal cells do not form tumors and appear to be less immunogenic when transplanted. Compared to mesenchymal cells to date, fetal cells do not require feeder layers for growth or specific growth factors for differentiation. A single organ donation can create a consistent seed cell bank (MCB) that can be used for hundreds of thousands of patient treatments. For this fully defined and consistent cell bank, the safety of any potential virus or pathogen can be easily and simultaneously evaluated in tandem with the original organ donations that have been completed for serological and pathogenic analysis.

Primary cultures of differentiated fetal cells from specific tissues (such as cartilage, tendons, and skin) will determine the quality of the clinical cell bank developed, including chondrocytes (chondrocyte-derived progenitor cells), skin fibers Blast cell-derived progenitor cells, and specific cell sources of tendon-derived progenitor cells can develop from that particular tissue. The idea that the initial treatment of the tissue is important for the physiological properties of the cells produced later is accepted. The conventional technique in primary culture is to digest small pieces of tissue with enzymes to release all living cells for cell culture. This technique is particularly needed for "hard" tissues, but it is also used for soft tissues, and conventionally uses multiple digestion steps. In this way, intact different cell populations can be released and cultured in primary and secondary cultures (Carrascosa A, Audi L, Ballabriga A Pediatric Research 19: 720-727, 1985; Roche S et al, Biomaterials 22 : 9-18, 2001; Bae H. et al., The Spine Journal Vol. 8, No. 5, pages 92S-93S, 2008; Reginato AMet al., Arthritis and Rheumatism, Vol. 37, No. 9 ,, 1994).

However, it is known that the enzymatic treatment will cause inconsistencies, resulting in different cell populations with different appearance, physiological properties, stability and functions (Diaz-Romero J, Gaillard JP, Grogan P, Nesic, Trub T, Varlet PM J Cellular Physiol 202 : 731-742, 2005).

The articular cartilage, due to its non-vascular, nerve-free, and lymphatic-free nature, makes repairing this tissue a challenge for both surgeons and tissue engineers. As the gold standard for osteochondral treatment strategies, especially the choice of cell source, it remains a central and controversial topic. No decision yet. To date, adult mesenchymal stromal cells (MSCs) are still the most commonly used cell source, even considering the phenotype's homogeneity, reliability, and stability.

Because there is still no satisfactory treatment solution for bone and joint defects, the treatment of it needs improvement. In particular, it is particularly important to develop new solutions to avoid the immature degradation of cartilage to avoid replacing the entire joint. The development of new cell-assisted surgery technology is based on a limited cell bank product that can meet the requirements of strict therapeutic agent preparation.

Therefore, there remains a need to develop methods for manufacturing new tissues such as tendons, cartilage, other musculoskeletal tissues, and skin for use in therapeutic strategies. More specifically, there is a need for a cell bank that provides a more stable and homogeneous population of cells and to find an appropriate source of cells that does not risk triggering an immune response and does not carry any infectious agents.

In order to solve the above-mentioned problems, the applicant has established a non-enzymatic method, which is a group of early attached cells that physically and quickly defines the characteristics of establishing a parent cell bank (PCB). In some specific examples, the primary, differentiated cell line is derived from specific cartilage tissue, specific tendon tissue, and specific skin tissue. For those skilled in the art, other specific examples may include primary differentiated cells from skin and musculoskeletal tissues, such as tendons, bones, muscles, and intervertebral discs. Developing a PCB can complete further consistent and stable cell bank production.

Specifically, one embodiment of the present invention provides an in vitro non-enzymatic method for isolation, expansion and development selected from the group consisting of fetal epiphyseal chondrocytes, fetal Achilles tendon cells, or fetal skin fibroblasts. Fetal cells, including the following steps: a) using a fetal sample selected from fetal ulnar cartilage containing fetal epiphyseal chondrocytes; fetal Achilles tendon containing fetal Achilles tendon cells; or fetal abdominal skin containing fetal skin fibroblasts B) microsectioning the fetal ulnar cartilage, fetal Achilles tendon or fetal abdominal skin samples and dispersing by physically attaching to a scalpel-scribed surface; c) using the fetal epiphyseal chondrocytes, fetus in vitro Achilles tendon cells or fetal abdomen skin fibroblasts proliferate under conditions of culture of the fetal ulnar cartilage, fetal Achilles tendon, or fetal abdominal skin samples; d) select and isolate the earliest fetal epiphyseal chondrocyte cell population and earliest attachment Fetal Achilles tendon cell population, and the earliest attached fetal skin fibroblast cell population.

In another specific example, the present invention provides fetal cells, which are obtained by the non-enzymatic method of the present invention. The fetal cells, such as: fetal epiphyseal chondrocyte cell line, are named FE002-Cart and registered as BCRC 960459; fetal Achilles tendon The cell line was named FE002-Ten and registered as BCRC 960461; the fetal skin fibroblast cell line was named FE002-SK2 and registered as BCRC 960460.

In another specific example, the present invention provides the use of fetal cells obtained by the method of the present invention, by using the differentiated cartilage, tendon or skin cells integrated into various substrates to create new cartilage tissue and / or three-dimensional Construct, new tendon tissue and / or three-dimensional construct, new skin tissue and / or three-dimensional construct.

In another specific example, the present invention provides fetal cells obtained by the method of the present invention as a therapeutic agent.

In another specific example, the present invention provides fetal cells obtained by the method of the present invention, which are used for repairing and regenerating osteochondral tissue and musculoskeletal tissue, and repairing and regenerating tendon tissue and musculoskeletal tissue. And methods of repairing and regenerating skin tissue, and treating burns, trauma, and fibrosis.

In another specific example, the present invention provides fetal cells obtained by the method of the present invention, which are used for treating osteochondral diseases or defects, arthritis, and musculoskeletal diseases; for treating musculoskeletal diseases and tendon diseases Method; used in the treatment of skin diseases.

In yet another embodiment of the present invention, a screening method is provided for the development of therapeutic agents and / or medicine for the treatment of arthritis, osteochondral defects, cartilage repair, tendon repair, musculoskeletal tissue repair, and skin repair. A device comprising fetal epiphyseal chondrocytes (FEC), fetal Achilles tendon cells, or fetal skin fibroblast cells obtained using the non-enzymatic method of the present invention.

Figure 1 shows the biopsy of the parental cell bank directed at fetal skin-derived progenitor cells. Cell selection on day 4 showed consistency.

Figure 2 shows the growth of fetal skin-derived progenitor cells after low-density inoculation (~ 2000 cells / cm ² ), and the recovery of frozen reserve cells on days 6 and 12 shows high stability, consistency, and maintenance function .

Figure 3 shows the differences in cell population selection and growth under conditions in which the tissue is enzymatically digested to prepare a parental cell bank, and the cell populations are inconsistent.

Figure 4 shows the biopsy of the parental cell bank directed to fetal tendon-derived progenitor cells (fetal Achilles tendon cells). Cell selection on day 4 shows consistency.

Figure 5 shows the growth of fetal tendon-derived progenitor cells after low-density inoculation (~ 2000 cells / cm ² ), and the recovery of frozen reserve cells on days 0, 3, 7 and 10 shows high stability and consistency. Sexual and functional.

Figure 6 shows FACS analysis and 3D matrix deposition characteristics for fetal tendon-derived progenitor cells.

Figure 7 shows the treatment of the epiphyseal tissue of the proximal ulna to proceed with the production of a parental cell bank directed against fetal cartilage-derived progenitor cells (fetal epiphyseal chondrocytes). Cell selection on day 6 showed consistency.

Figure 8 shows the growth of fetal cartilage-derived progenitor cells (fetal epiphyseal chondrocytes) after low-density inoculation (~ 2000 cells / cm ² ), and the recovery of frozen reserve cells on days 6 and 12 shows high stability , Consistency and maintain function.

Figure 9 shows FACS analysis and 3D matrix deposition characteristics for fetal cartilage-derived progenitor cells (fetal epiphyseal chondrocytes).

Figure 10 shows the expansion of fetal cell-derived progenitor cells on a tissue culture dish along a physically scored surface.

Figure 11 shows the rapid development of clinical parental cell banks using non-enzymatic preparation of tissues.

Although methods or materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the present invention still hereinafter describes suitable methods and materials. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The publications and applications discussed herein are provided solely for matters disclosed before the filing date of the present invention. It should not be interpreted as an admission that the present invention cannot be made earlier than the publication by virtue of an earlier invention date. In addition, the materials, methods, and examples are for ease of understanding only and are not intended to be limiting.

In case of conflict, including definitions, this specification shall prevail. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this subject belongs. Definitions are provided below to assist in understanding the invention.

As used herein, "a" or "an" means "at least one" or "one or more".

The term "comprising" is generally used in the sense of inclusion, meaning that one or more features or components are allowed.

The term "biological material" means natural or synthetic materials, including when in contact with cells or biological tissues No harmful metals, ceramics and polymers. In general, biomaterial support systems are selected from the group consisting of polymerizable supports including olefin polymers, fluoropolymers, polystyrene, polyacrylic polymers, polyester polymers, polyurethane polymers, silicon polymers, and fibers. Element polymers, epoxy polymers, silicone polymers, synthetic hydrogels, polycarbonates; biocompatible metal supports, including: titanium and titanium alloys, nitinol, Zirconia, stainless steel and cobalt chromium, alumina-zirconia composites; and / or biocompatible ceramics, including: porcelain, hydroxyapatite, and mixtures thereof.

The terms "fetal chondrocytes or cartilage-derived progenitor cells", "fetal Achilles tendon cells or fetal tendon-derived progenitor cells", or "fetal fibroblasts or fetal skin-derived progenitor cells" are compared to undifferentiated fetal cells. Refers to differentiated cells. In contrast to the present invention, the term "undifferentiated" is used to describe immature or primitive cells. For example, undifferentiated fetal skin cells include those that can differentiate into dermal fibroblasts, epidermal keratinocytes, or even other specific cell types that are not related to tissue. Differentiated cells do not de-differentiate into different cell lineages when placed in a differentiation medium or different microenvironment specifically for other cell types. For example, if fetal skin fibroblasts are placed in osteogenic differentiation medium, they will not become a complete osteoblast population, or if they are produced in adipocyte-forming medium, they will not become completed adipose populations, and If the same cells are placed on a 3D matrix connected to bones, they will not be redifferentiated into a complete osteoblast population due to environmental changes. These defined, differentiated cells have important advantages in terms of their potential use as therapeutic agents for human and animal medicine.

The term "appropriate culture conditions" or "conditions for the proliferation of fetal epiphyseal chondrocytes or chondrocyte-derived progenitor cells, fetal tendon-derived progenitor cells, or fetal skin-derived progenitor cells" is a medium for culturing cells, which contains a Nutrients. The nutrient medium may contain an appropriate combination and concentration of any of the following components: isotonic saline solution, buffer solution, amino acid, serum or serum substitute, and other exogenous additive factors. Those of ordinary skill in the art will recognize that all commonly used culture conditions can be used. Techniques for selecting the most appropriate medium, medium preparation, and cell culture are well known in the art and have been described in many literatures, including Doyle et al., (Eds.), 1995, Cell & Tissue Culture: Laboratory Procedures, John Wiley & Sons, Chichester; and Ho and Wang (eds.), 1991, Animal Cell Bioreactors, Butterworth-Heinemann, Boston, which are incorporated herein by reference. For example, any suitable type of culture medium can be used to isolate fetal epiphyseal chondrocytes of the present invention, such as but not limited to In DMEM, McCoys 5A medium (Gibco), Eagle's basal medium, CMRL medium, Glasgow minimally necessary medium, Ham's F-12 medium, Iscove's modified Dulbecco's medium, Liebovitz 'L-15 medium, RPMI 1640, serum-free medium and so on. The medium may be supplemented with one or more ingredients including, for example, fetal bovine serum (FBS), defined growth factor serum substitutes, horse serum (ES), human serum (HS), defined cell culture growth factors, and a Or more antibiotics and / or antifungals for controlling microbial contamination, such as penicillin G, streptomycin sulfate, amphotericin B, gentamicin, and nystatin ), Can be alone or in combination.

The term "cell line" refers to a permanently established cell culture that will proliferate indefinitely when given an appropriate fresh medium and sufficient space. The term primary cell line refers to an established cell line with a limited number of passages.

The term "cell bank" refers to: collecting biopsy tissue from donor fetal tissues, such as fetal ulna cartilage, fetal Achilles tendon, or fetal skin; growing the fetal tissue under appropriate culture conditions and proliferating the fetal cells to a high concentration; Treat the tissues and cells of the final culture with an enzymatic or non-enzymatic treatment (ie, trypsin) to suspend them; assemble the suspended cells to make a roughly uniform suspension of cell lines from the culture; Carefully mix with an antifreeze protector; seal a sample of the cell suspension in an ampoule; freeze the samples. The optimum freezing speed can be determined experimentally. For example, the temperature of the ampoule is reduced to -80 ° C at 1 ° C / min, and then converted to -160 ° C after about 24 hours, or frozen in a calibrated Nano-Freezer in a stylized cycle to complete the cycle to- 165 ° C. This ultra-cold temperature library can save cells from aging and maintain their function and activity on the day of collection.

The cryopreserved cells of the present invention constitute a cell bank (1-10 ⁷ / ml), a part of which can be "withdrawn" by thawing, and then used to make the required new chondrocytes and tissues. Thawing should generally be performed quickly, for example, by moving an ampoule taken from liquid nitrogen to a 37 ° C water bath. The thawed content in the ampoule should be immediately transferred under sterile conditions to a culture vessel containing an appropriate medium such as DMEM formulated with 10% FBS. It is recommended to adjust the initial cell density in the culture medium to approximately 1-6 × 10 ^3-4 cells / ml. During culture, cells can be inspected daily, for example, by using an inverted microscope to detect cell proliferation, and subcultured when the cells reach an appropriate concentration, or monitored by a scanning microscope for quality control.

If desired, the cells of the invention can be extracted from the cell bank and formatted as described herein. Cartilage, tendon, or skin culture in the form of new cartilage, tendon, or skin tissue or cells in vitro, or by in vitro, such as the direct administration of cells to a subject, requires new cartilage, new tendon, or new skin tissue or That part of the cell.

The term "subject" ("subject" as a treatment) or "patient" means a mammalian individual who is afflicted with a disorder, defect, or disease (as specified herein), or who is prone to or has become ill. This term does not indicate a specific age or gender. Therefore, both adult and newborn subjects, male or female, are intended to be covered. The term also includes humans and animals. For example, the object may be, for example, a human, a primate other than a human, a wild animal, a dog, a cat, a horse, a cow, a pig, a sheep, a goat, a rabbit, a rat, or a mouse. People and horses are better candidates. As used herein, the term wildlife includes non-domesticated mammals, birds, secondaries, or fish. Such wild animals are, for example, but not limited to, cormorants, beavers, lions, tigers, bears, eagles and deer.

Stereoscopic matrix means any matrix selected from collagen matrix or PLA, PLGA, PEG, chitosan, elastin, hydrogel including, for example, HA (hyaluronic acid), silicone, chitosan or mixtures thereof . The matrix provides a three-dimensional space to ensure proper coverage, and transfers fetal cells such as fetal epiphyseal chondrocytes or cartilage-derived progenitor cells, fetal tendon-derived progenitor cells, fetal skin-derived progenitor cells, or fetal products to or with additional Migration materials are transmitted together. In a specific example, the method of the present invention allows the use of differentiated cartilage, tendon, or skin cells integrated into various matrices to prepare a three-dimensional structure. Integration of the differentiated cartilage, tendon, or skin cells with the matrix can occur by mixing, combining, and pipetting the cells with the matrix, plating, covering, or placing the cells on the matrix.

The term "collagen" refers to a polypeptide compound, which is hydrophilic in nature and is susceptible to degradation by extracellular enzymes. Because the substance has been studied transparently, it can control many key parameters. Collagen is a weak antigen, so it is extremely unlikely to cause rejection. The preferred collagen used in the methods and uses of the present invention is horse collagen or or porcine collagen.

An "imPlant" can be regarded as a medical device, which is to replace a missing biological structure, support a damaged biological structure, or enhance an existing biological structure. Implants can be made from artificial or natural materials. Medical implants are artificial devices, and the surface of the implant that contacts the body can be made of biomedical materials, such as titanium, silicone, polymers, apatite, biofoam, and biogels. Some implants can have a combined bioactive eluent, such as an implantable capsule or a drug eluent guide. The implant material can be combined with differentiated fetal cells of a particular tissue type to respond to an anti-fibrotic response.

The term "delivery system" refers to any metal that provides the delivery of fetal cells or fetal cell products, alone or with implants, to process tissues or to implement implantation mechanisms, or orthopedics, trauma, maxillofacial natural or synthetic, which are customarily used Implant material, hydrogel, silicone or graft.

The term "cartilage tissue" as used herein is a term generally recognized in the art and refers to a specialized type of dense connective tissue including cells embedded in the ECM (see, for example, Cormack, 1987, Ham's Histology , 9th Ed., JBLippincott Co., pp. 266-272). The biochemical composition of cartilage varies by type; however, the general composition of cartilage includes chondrocytes surrounded by a dense ECM composed of collagen, proteoglycans, and water. Several types of cartilage have been recognized in this technical field, including, for example, hyaline cartilage, articular cartilage, rib cartilage, fibrocartilage, meniscal cartilage, elastic cartilage, ear cartilage, and yellow cartilage. The preparation of any cartilage type is intended to be included within the scope of the present invention. According to a specific example, the present invention indicates a method and a composition for producing new cartilage tissue for human use. However, the present invention can also be implemented to produce new cartilage tissues for use in any mammal in need, including horses, dogs, cats, sheep, pigs, and the like. The treatment of such animals is also intended to be encompassed within the scope of the present invention.

The term "tendon tissue" as used herein is a term generally recognized in the technical field, and refers to a specialized type of fibrous connective tissue mainly comprising collagen, proteoglycan, and water.

The term "skin tissue" as used herein is a term generally recognized in the technical field and refers to a fibrous tissue of a specialized type that also includes collagen, elastin, and proteoglycans.

In a specific example of the present invention, a non-enzymatic method is disclosed, which uses physical and rapid release to define an early population of attached cells that establishes the characteristics of a parental cell bank (PCB). In some specific examples, the primary, differentiated cells are derived from specific cartilage tissue, from specific tendon tissue, and from specific skin tissue. The development of PCB can achieve the consistency and stability of subsequent cell bank manufacturing.

Therefore, according to a specific example of the present invention, an in vitro non-enzymatic method is provided for isolation, expansion and development selected from the group consisting of fetal epiphyseal chondrocytes, fetal Achilles tendon cells, or fetal skin fibroblasts. Fetal cells, including the following steps: a) using a fetal sample selected from fetal ulnar cartilage containing fetal epiphyseal chondrocytes; fetal Achilles tendon containing fetal Achilles tendon cells; or fetal abdominal skin containing fetal skin fibroblasts ; B) microsectioning the fetal ulnar cartilage, fetal Achilles tendon, or fetal abdominal skin samples, and Physically adhere to the surface scribed by a scalpel to disperse it; c) culture the fetal ulnar cartilage, fetal heel in vitro under conditions of proliferation of the fetal epiphyseal chondrocytes, fetal Achilles tendon cells, or fetal abdominal skin fibroblasts Tendon or fetal belly skin samples; d) Select and isolate the earliest attached fetal epiphyseal chondrocyte cell population, the earliest attached fetal Achilles tendon cell population, and the earliest attached fetal skin fibroblast cell population.

The ideal fetal ulnar cartilage sample is a fetal proximal ulna callus.

In a specific embodiment, the present invention relates to the isolation, expansion and development of fetal epiphyseal chondrocytes or chondrogenic progenitor cells (FEC), fetal Achilles tendon-derived progenitor cells from a single tissue donor (only 0.2-2 cm tissue). , And method of parental cell bank of fetal skin-derived progenitor cells. Sources of cartilage, tendons, and skin are important for building a consistent cell bank. The applicant has found that fetal ulnar cartilage is a source superior to femur, tibia or rib cartilage, fetal Achilles tendon is ideal, and skin from the fetal abdomen is ideal. Fetal cartilage, tendon, and skin have excellent ability to repair, and fetal cells show immunomodulatory activity and significant wound healing ability. For cartilage, in this respect, it is combined with its natural osteochondrogenesis ability after epiphyseal osteogenesis, and the cell characteristics of FEC, so that the cell population isolated by the non-enzymatic method of the present invention becomes used for osteochondral and / Or cartilage tissue repair and regeneration is a very interesting cellular option.

The method of the present invention does not use a conventional method for digesting primary cultures for releasing a selected cell population. Digestion is usually applied to tissues that do not break down automatically (ie, blood cell components, some placental tissue parts, some umbilical cord parts). The present invention combines physical slicing of tissues and guides tissues / cells to grow into the tissue culture plate along the score. Only the earliest attached cell population is selected after a few days of growth, which is very consistent and homogeneous. These cell populations grow faster and have different shapes and physiological conditions than other cell populations digested with enzymes. The applicant has presented a two-dimensional growth difference between fetal skin tissues digested with enzymes and fetal skin tissues that are physically treated with cells arranged on the surface.

It is important that if the primary culture is developed without tissue digestion, chondrocytes, fetal tendon-derived progenitor cells, and fetal skin-derived progenitor cells of the fetal joints will not easily dedifferentiate into other cell lines. Chondrocytes or cartilage-derived progenitor cells, tendon-derived progenitor cells, and skin-derived progenitor cells that have not undergone enzyme digestion during the process of making cell banks do not differentiate into nerves, fat-forming, and intact osteoblasts (such as Mesenchymal stem cells), but these cells have bone marrow that has been treated with enzymes from their primary cell culture or does not have solid tissue components for attachment to plastic culture dishes Or derived from chondrocytes of fetal joints. Another important point is that cell banks developed from primary cell cultures of non-enzymatic cells have more stable cell passage in vitro. For human therapeutics using cells made into such cell banks, the appearance and stability of chromosomes are important factors.

A non-enzymatic method and a complete cGMP isolation method for observing the homogeneity and stability of the FEC population, tendon-derived progenitor cell population, and skin-derived progenitor cell population in culture are adopted, which can allow the in vitro use of this method to have Reliably expand FEC, tendon-derived progenitor cells, and skin-derived progenitor cells, and develop a large number of seed cell banks from the PCB population. Moreover, the same cell bank of various tissues can also be used for clinical applications of hundreds of thousands of patients, opening a door for novel osteochondral, musculoskeletal, and skin regeneration therapies.

Musculoskeletal tissues such as tendons, bones, muscles, discs, and cartilage, as well as skin tissues derived from fetal tissue at 10-16 weeks are rapidly developing as an ideal source of parental cell banks for clinical use. If the tissue is not digested with enzymes and the cell line is guided along the jagged surface, the cells from it can be quickly prepared (within 12-14 days, compared with the enzyme-digested tissues for several weeks to For several months), its population is homogeneous and it maintains specific tissue-to-cell types with associated biomarkers.

The non-enzymatic method of the present invention provides cells different from those obtained by known enzyme methods. Therefore, in a specific example, the present invention provides a fetal epiphyseal chondrocyte cell line, which is obtained by the non-enzymatic method of the present invention, named FE002-Cart, and has been deposited on December 24, 2012 under the registration number BCRC 960459. In another specific example, the present invention provides a fetal Achilles tendon cell line (also described as a fetal Achilles tendon-derived progenitor cell line), which is obtained by the non-enzymatic method of the present invention and named FE002-Ten and has been in 2012. Deposited on December 24 with the deposit number BCRC 960461. In another specific example, the present invention provides a fetal skin fibroblast cell line (also described as a fetal skin-derived progenitor cell line), which is obtained by the non-enzymatic method of the present invention and named FE002-SK2 and has been in 2012. On December 24, it was deposited with the deposit number BCRC 960460.

Once a fetal epiphyseal chondrocyte or chondrogenic progenitor cell culture is established, it can be used to produce chondrocytes capable of producing new chondrocytes and tissues. The differentiation of fetal epiphyseal chondrocytes into chondrocytes and subsequent production of cartilage tissue can be triggered by the addition or absence of special exogenous growth factors to the culture medium, such as BMP with or without addition of ascorbate, such as BMP-13 Or TGF-β. Can be applied to fetal tendon-derived progenitor cells and fetal skin-derived progenitor cells The same procedure has been established by cells.

The invention further considers the establishment and maintenance of a culture of chondrocytes and a mixed culture comprising fetal epiphyseal chondrocytes and chondrocytes. For fetal epiphyseal chondrocytes, when a culture of chondrocytes, or a culture of fetal epiphyseal chondrocytes and chondrocytes is established, the cell population is changed to the cell culture medium depending on the cell density. Conditions that do not form cartilage, such as mitotic expansion in vitro in media lacking TGF-β or other growth factors. For chondrocyte-derived progenitor cells, it is necessary to transfer the culture of chondrocytes and the mixed culture of fetal epiphyseal chondrocytes and chondrocytes to fresh medium when sufficient cell density is reached. Therefore, the formation of a monolayer of cells should be prevented or minimized, and as a matter of practice, it can be carried out by, for example, moving a part of the cells to a new culture container or to a fresh medium. Such removal and transfer should be performed in any culture vessel in which the cell monolayer exceeds about 25% confluency. Alternatively, the culture line can be agitated to prevent cell sticking. This same method can also be applied to fetal tendon-derived progenitor cells and fetal skin-derived progenitor cells.

In another embodiment of the present invention, a group of fetal epiphyseal chondrocytes or cartilage-derived progenitor cells isolated from fetal ulnar cartilage is mitotically expanded and cultured in vitro to produce cartilage tissues and cells for therapeutic use. Chondrocytes. The same method can also be applied to fetal tendon-derived progenitor cells and fetal skin-derived progenitor cells. Therefore, in a specific example, the present invention relates to the use of fetal epiphyseal chondrocytes (FEC), fetal Achilles tendon cells, and fetal skin fibroblasts as therapeutic agents obtained by the non-enzymatic method of the present invention.

In another specific example of the present invention, fetal epiphyseal chondrocytes or cartilage-derived progenitor cell lines isolated from fetal ulnar cartilage are cryopreserved and stored frozen in a "bank", and can be thawed from the bank and used to make cartilage tissue and cell. The fetal epiphyseal chondrocytes or cartilage-derived progenitor cells collected or produced from this can be frozen in a "bank" for several years. The cells can be extracted from the library by thawing as needed, and the thawed cells can be used to make new tissues and cells for repair, replacement or lengthening of cartilage, and other mesenchymal tissues such as bones, tendons or ligaments. This method can be similarly applied to fetal tendon-derived progenitor cells and fetal skin-derived progenitor cells.

Because the cells isolated from the fetal ulnar cartilage sample have the "fetal" nature, the immune rejection of the transplanted fetal epiphyseal chondrocytes or cartilage tissue produced from them can be minimized. Therefore, according to another embodiment of the present invention, such a cell is useful as a "ubiquitous donor cell" system for use in any desired object. Same characteristics for fetal tendon-derived progenitor cells and Fetal skin-derived progenitor cells are also observed.

In another specific example of the present invention, fetal epiphyseal chondrocytes or cartilage-derived progenitor cells, fetal Achilles tendon-derived progenitor cells, or fetal skin-derived progenitor cells are suspended in a hydrogel solution to enable injection or transplantation into a patient. Alternatively, the cells can be seeded into a hydrogel and then cultured before transplantation. Preferably, the cells are cultured in a hydrogel so that they are expanded by mitosis before transplantation.

In yet another specific example of the present invention, a new cartilage tissue and cell population is prepared from fetal epiphyseal chondrocytes or chondrogenic progenitor cells of the present invention, and is used to repair, replace or grow any of Or future technologies to repair, replace, or grow cartilage tissue in a subject. For example, the fetal epiphyseal chondrocytes or cartilage-derived progenitor cells of the present invention can be seeded on a three-dimensional frame or scaffold made of non-living materials that are compatible with the living body. Void space, opening or hole. Under appropriate in vitro culture conditions, the seeded cells will substantially envelope the three-dimensional framework and secrete extracellular matrix to form new, living cartilage tissue that can be transplanted in vivo. Alternatively, the fetal epiphyseal chondrocytes or chondrogenic progenitor cells of the present invention are seeded in a three-dimensional frame and immediately transplanted to a part of the subject. The inoculated cells undergo new cartilage formation in the body, or stimulate the recipient's cartilage repair and reorganization. The method can also be applied to fetal tendon-derived progenitor cells and fetal skin-derived progenitor cells to form or stimulate new tendon tissue or new skin tissue repair or reorganization of the recipient's tendon or skin.

In another specific example, the three-dimensional frame in which the cells of the present invention are seeded further comprises or covers one or more biologically active agents or other groups selected from the group consisting of anti-inflammatory agents, growth factors, immune factors, and the like. Compounds in group.

In yet another specific example of the present invention, the fetal epiphyseal chondrocytes or cartilage-derived progenitor cell lines of the present invention are inoculated and grown on a three-dimensional frame, and placed in a container that can tolerate intermittent pressure changes, or placed in a special purpose To produce cartilage tissue structures in vitro, the bioreactor system allows the cavity to be pressurized during growth and provides sufficient nutrient replenishment to the chondrocytes by convection. The same method can also be applied to fetal tendon-derived progenitor cells and fetal skin-derived progenitor cells.

Scaffold-based cell therapy offers a very clear advantage, that the therapeutic tissue-generating agent delivered, in this case, a cell, can be easily positioned and can be transplanted with the stent by arthroscopic surgery (Iwasa et al. , 2009). This treatment bypass requires major invasive surgery (whole joint replacement) and preserves the original tissues as much as possible, so a significant reduction can significantly negatively affect the occurrence of inflammation in the treatment (van Osch et al. , 2009). In order to provide a template that supports 3D tissue growth, the structure and composition of the scaffold need to be carefully matched. Synthetic materials, such as polyethylene glycol (PEG), polylactic acid (PLA), and polyglycolic acid (PGA), and naturally derived materials, such as hyaluronic acid, chondroitin sulfate, and chitosan, have been further developed. The state with or without chemical modification and side groups is used to generate bone and cartilage (Ahmed et al. , 2010; Chung et al. , 2008; Khan et al. , 2008).

Biomaterial scaffolds function as an extracellular matrix to provide physical structures that protect cells and guide tissue growth. Integration into the matrix is necessary to have a standing system for delivery to the surgical site of interest. Fetal cells, unlike adult and mesenchymal cells, are shown to penetrate various biological materials due to their inherent attachment and migration properties. This property allows the cells to be quickly seeded into their respective biomaterials and requires less treatment of the graft before transplantation. Different materials and construction methods can form biomaterials with different properties that are suitable for cartilage tissue engineering. Many biodegradable biomaterials and hydrogels have been developed for other surgical uses, such as hemostatic sponges and tissue fillers (Mast et al. , 1993; Patino et al. 2002; Drury and Moony, 2003). These biomaterials provide clinical grade materials (classified as medical devices) that need to be tested for biocompatibility and ensure that there are no radioactively derived products due to the biomaterial delivery system and the cellular product related.

In another specific example, the fetal epiphyseal chondrocytes or cartilage-derived progenitor cells of the present invention are not attached to a three-dimensional frame, but are directly injected into a part of the body by, for example, injection to generate new cartilage tissue in the part. And cell populations. The same method can also be applied to fetal tendon-derived progenitor cells and fetal skin-derived progenitor cells. Therefore, in a specific example, the present invention relates to the use of fetal epiphyseal chondrocytes (FEC) obtained by the non-enzymatic method of the present invention, which is used for manufacturing new cartilage tissue and / or three-dimensional structures; The use of fetal Achilles tendon cells obtained by the enzyme method is used for manufacturing new tendon tissue and / or three-dimensional structure, and the use of fetal skin fibroblasts obtained by the non-enzymatic method of the present invention is used for manufacturing new Skin tissue and / or three-dimensional structures.

In another specific example of the present invention, the fetal epiphyseal chondrocytes or chondrogenic progenitor cells of the present invention Cells are stimulated with exogenously supplied growth factors such as TGF-β, or BMPs such as BMP-2, BMP-12 and BMP-13 to produce cartilage.

In yet another specific example of the present invention, the fetal epiphyseal chondrocytes or chondrogenic progenitor cells of the present invention may be genetically engineered to produce or increase specific growth factors, peptides, proteins, or other actions that enhance the amount of cartilage produced, Or improve the success rate of the transplant, such as molecules that reduce the risk of rejection or accompany inflammation of the graft or increase yield. The same method can also be applied to fetal tendon-derived progenitor cells and fetal skin-derived progenitor cells.

The present invention also relates to the products of the foregoing methods, including but not limited to fetal epiphyseal chondrocytes or chondrogenic progenitor cells of the present invention, mitotic amplifiers, etc .; new cartilage tissue produced therefrom; Outer matrix; and stereo cartilage / framework. The present invention also relates to the use of these cells, structures and tissues for repairing, replacing or growing cartilage in vivo, or forming a three-dimensional cartilage culture in vitro for producing new cartilage tissue or a biologically active agent, Or test the cytotoxicity of potential therapeutics. The same method can also be applied to fetal tendon-derived progenitor cells and fetal skin-derived progenitor cells.

In a specific example of the present invention, fetal epiphyseal chondrocytes or cartilage-derived progenitor cells isolated from fetal ulnar cartilage and chondrocytes differentiated therefrom can be used to produce new cartilage tissues and cells for repair or replacement of cartilage. The same method can also be applied to fetal tendon-derived progenitor cells and fetal skin-derived progenitor cells. These can be used to produce new tendons or skin tissues and cells for repair or replacement of tendons or skin.

In another embodiment of the present invention, fetal epiphyseal chondrocytes or chondrogenic progenitor cells isolated from fetal ulnar cartilage according to the method of the present invention and chondrocytes differentiated therefrom can be used as a therapeutic agent. Preferably, fetal epiphyseal chondrocytes isolated from fetal ulnar cartilage and chondrocytes differentiated therefrom according to the method of the present invention are used for osteochondral repair (including cartilage repair and tendon repair) and regeneration methods and for treatment. Methods of osteochondral disease or defect (including osteochondral lesions, injuries, trauma, fragmentation and fractures, osteonecrosis of the lower cartilage and osteochondritis) and / or arthritis. The same method can also be applied to fetal tendon-derived progenitor cells and fetal skin-derived progenitor cells for specific treatment of tendons and skin.

Therefore, in a specific example, the present invention relates to a fetal epiphyseal chondrocyte (FEC) obtained by the non-enzymatic method of the present invention, which is a method for repair and regeneration of osteochondral tissue and musculoskeletal tissue; Fetal Achilles tendon cells obtained by non-enzymatic methods, which are methods for repairing and regenerating tendon tissue and musculoskeletal tissue; and a non-enzymatic method obtained by the present invention Fetal skin fibroblasts are methods for repairing and regenerating skin tissue, and for treating burns, wounds, and fibrotic conditions.

In another specific example of the present invention, there is provided: fetal epiphyseal chondrocytes (FEC) obtained by the non-enzymatic method of the present invention, which is used for treating osteochondral diseases or defects, arthritis, and musculoskeletal diseases; a Fetal Achilles tendon cells obtained by the non-enzymatic method of the present invention are used for the treatment of musculoskeletal diseases and tendon diseases; a fetal skin fibroblast cell obtained by the non-enzymatic methods of the present invention are used for the treatment of skin Methods of disease.

The use of fetal epiphyseal chondrocytes (FEC) or chondrogenic progenitor cells in osteochondral tissue engineering is very promising. In fact, throughout the development of the fetus, this epiphysis becomes the main site of the secondary ossification center (SOC), which transforms FEC condensate into the area of cartilage of the joint, and before the epiphyseal cord after blood vessel invasion Trabecular bone and bone marrow (Blumer et al. , 2008; Onyekwelu et al. , 2009). It is known that fetal cartilage, as observed in fetal skin, has a strong ability to repair itself. In a goat fetal model, it was shown that the defect was repaired without lemma or fibrous tissue formation (Namba et al. , 1998).

The cells of the present invention (fetal epiphyseal chondrocytes or chondrogenic progenitor cells and chondrocytes) and cartilage tissue can be used in vitro to screen a variety of compounds for the effectiveness and cytotoxicity of pharmaceutical agents, growth / regulatory factors, anti-inflammatory agents, and the like. To this end, the cells, or tissue cultures of the invention as described above, are maintained in vitro and exposed to the compound to be tested. The activity of a cytotoxic compound can be measured by its ability to damage or kill cells in culture. It can be easily evaluated using live staining techniques. The effects of growth / regulatory factors can be assessed by analyzing the number of living cells in vitro, such as total cell counts, and counting the number of cells that differ. This can be achieved by using standard cytological and / or histological techniques, including immunocytochemical techniques using antibodies that recognize type-specific cellular antigens. The effects of various drugs on the cells of the present invention in suspension culture or the above-mentioned state of the system can be evaluated. Therefore, fetal epiphyseal chondrocytes or cartilage-derived progenitor cells isolated from fetal ulnar cartilage can also be used to develop advanced therapeutic agents and / or medical devices for the treatment of arthritis, osteochondral defects, cartilage repair, and tendon repair. The same method can also be applied to fetal tendon-derived progenitor cells and fetal skin-derived progenitor cells, and it can also be applied to the development of advanced therapeutic agents and / or medical devices for treating musculoskeletal tissues.

Therefore, in one embodiment of the present invention, a screening method is provided for the development of osteochondral defects, cartilage repair, tendon repair, musculoskeletal tissue repair, and skin for the treatment of arthritis. Repairing therapeutic agents and / or medical devices include fetal epiphyseal chondrocytes (FEC), fetal Achilles tendon cells, or fetal skin fibroblasts obtained using the non-enzymatic method of the present invention.

The cells of the present invention (fetal epiphyseal chondrocytes or chondrogenic progenitor cells and chondrocytes) and cartilage tissue can be used as a model line for studying physiological conditions or pathogenic conditions. For example, fixed joints can be damaged fairly quickly in many ways. The metabolic activity of chondrocytes is affected by the loss of proteoglycans, and an increase in water content is quickly observed. The normal white, shiny appearance of cartilage becomes dull, the hue is bluish, and the thickness of cartilage is reduced. However, it is unknown to this day that the magnitude of this change is due to nutritional deficiencies and constant disruption of stress-dependent metabolism. The cells and cartilage tissue of the present invention can be used to determine the nutritional requirements of cartilage under different physical conditions, such as intermittent pressurization, and by driving nutrient medium into and out of cartilage structures. This is particularly useful for investigating the underlying causes of reduced tensile strength of articular cartilage (such as in the knee) related to age or injury, which makes weakened cartilage vulnerable to trauma. The same method can also be applied to fetal tendon-derived progenitor cells and fetal skin-derived progenitor cells. It is used to study the underlying causes of traumatic or age-related damage caused by the reduction of the tensile strength of tendons and skin related to injury or injury.

The cells (fetal epiphyseal chondrocytes or chondrogenic progenitor cells and chondrocytes) and cartilage tissue of the present invention can also be used to study the mechanism of action of cytokines and other pro-inflammatory mediators such as IL-1, TNF and prostaglandins. Released into synovial fluid due to rheumatic diseases. Therefore, the patient's own joints can be tested in vitro to study the effect of these compounds on the growth of the cells of the invention. In addition, the most effective cytotoxic and / or pharmaceutical preparations can be screened for specific patients, such as those that reduce or prevent reabsorption of cartilage or otherwise promote balanced growth of articular cartilage. Agents that have proven effective in vitro can then be used to treat patients therapeutically. The same method can be applied to fetal tendon-derived progenitor cells and fetal skin-derived progenitor cells in the same way. It has been used to study the mechanism of growth factors, cytokines, and other inflammatory precursor mediators that cause imbalance in tissue repair.

Those having ordinary knowledge in the technical field to which the present invention pertains can understand that the present invention described herein may be changed and modified in addition to the special description. It should be understood that the invention includes all such alterations and modifications without departing from the spirit or essential characteristics of the invention. The present invention also includes all of the steps, characteristics, combinations, and compounds mentioned individually or collectively in this specification, and all combinations or any two or more of those steps or characteristics. Therefore, this disclosure is intended to be understood rather than limiting, and the scope of the present invention is specified by the scope of the accompanying patent application, and all All changes within the scope are intended to be included.

The foregoing description will be more fully understood from the following examples. However, these embodiments are exemplary methods implemented by the present invention and are not intended to limit the scope of the present invention.

[Example]

The epiphysis, Achilles tendon, and abdominal skin of the proximal ulna were processed in accordance with strict transplantation laws and detailed rules and guidelines for organ donation and screening to create a FEC, tendon, and skin parental cell bank for tissue engineering applications (CHUV Ethics Committee protocol # 62/07). Tissue biopsy sections (cartilage, ~ 2mm ³ , tendon, ~ 0.2mm ³ , skin, ~ 2cm ² ) are micro-sectioned and dispersed by physical attachment to a scalpel-scratched surface (without enzyme treatment, to Make sure that only the attached cartilage, tendon, or skin grows, and that the cell population is consistent: this line is a necessary criterion for clinical use). FEC, tendon, and skin growth were observed from 1 to 2 days to 1 week, and expansion was completed at 1 and 2 weeks. From 5-10 x 10 ⁶ cells per vial to 50-200 vials, the parental cell bank was established and stored in liquid nitrogen gas phase. Characterization of isolated cells in vitro showed excellent homogeneity in the form of monolayer cultures and significant proliferative potential (3D cultures) that tend to overlap. FEC, tendon-derived progenitor cells, and skin-derived progenitor cells did not appear to show significant phenotypic changes in the first 6 passages. FEC, tendon-derived progenitor cells, when placed in a pellet culture format, can spontaneously join, precipitate the matrix, and display basic chondrogenic or tendon cell marks. Flow cytometry analysis showed a unimodal distribution, representing a homogeneous population. Compared with MSCs derived from adult bone marrow, the results show that the contours of FEC or tendon surface markers are consistent with cartilage or tendon cells instead of the undifferentiated source progenitor phenotype.

Preparation of fetal chondrocytes, tendon-derived progenitor cells, and skin-derived progenitor cell banks, and characterization of the preparation of therapeutic agents

Making a Cell Bank

The cell bank department was established at the Lausanne University Hospital. It is a local medical school ethics committee that has been approved in writing and has been established since 1993. More specifically, it has been approved by the clinical cell bank established before 2007 to terminate pregnancy. Fetal biopsies obtained between 12 and 14 weeks of pregnancy. To date, preclinical articular chondrocyte cell banks have been successfully developed from 2 independent donors. Using one of them (FE002-Cart Generation 0; FE-002-Ten Generation 0; FE-002-SK2 Generation 0) will enable the establishment of all low-passage cells for preclinical and clinical trials (Section MCB of 3rd generation, WCB of 5th generation).

Pre-clinical cell banks of generations 0 and 1 were prepared from cartilage (radius) of about 0.3 cm ³ joints (see Fig. 7), Achilles tendon of about 0.2 mm ³ and abdominal skin of about 1 cm ² . Tissues were cut into pieces as small as <0.5mm ³ as much as possible to grow in DMEM medium supplemented with 10% FCS and glutamic acid, and the cells were used for qualitative and experimental purposes at passages 3 and 6 or passages 1 and 9 . They were grown to confluence before cutting and washed twice with PBS and counted.

Detailed procedure without enzyme treatment: As far as possible, divide the tissue into two 10cm flat disks, with about 5 complete tissue fragments (<0.5mm ³ ) per disk. First, the tissue culture dish was deeply drawn into a checkerboard pattern with a sterile scalpel under a laminar flow hood. Place fragments of the tissue into the plastic area where it is depicted, gently shred and attach the fragments to the plastic cuts. Place a small amount of nutrient medium around each fragment to prevent the tissue from floating in the first 24 hours. After the first 24 hours, 8 ml of medium was added to each 10 cm flat plate and changed twice a week before passage. These fragments were grown in DMEM supplemented with only 10% fetal bovine serum (Hyclone) to help ensure the consistency of the cell culture. Cell cultures were grown at 37 ° C in a humidified atmosphere of 95% air / 10% carbon dioxide. Important and need to mention: All nutritional components necessary for clinical trials of cell cultures should have complete safety requirements and tracking. All animal-derived products, such as fetal bovine serum and trypsin, should use specific clinical batches of trypsin and gamma-irradiated serum that have been tested for foreign agents. Although cell growth can be observed after day 1, tissues and cell dishes are trypsinized (0.25% trypsin-0.1% ethylene diaminetetraacetic acid [EDTA]) after 5-7 days of cell regrowth or used alone EDTA is removed from the plate for passage into multi-tissue culture flasks or frozen for cell banks. At this point, some fetal cartilage, fetal tendon, or fetal skin cells are frozen in liquid nitrogen in individual units, and others are passaged at 1,000-2,000 cells / cm ² or 10,000-50,000 cells / cm ² for production.制 PCB。 System PCB. The cells were centrifuged at 2000 g for 15 min and resuspended in a frozen solution of DMEM (5 ml) + FCS (4 ml) + DMSO (1 ml, Fluka), frozen at -80 ° C, and 1 ml aliquots in a Nalgene Cryo 1 ° C freezer (Nalgene). (~ 5-10 million cells) reached 1 ° C / min cooling and freezing curve. After 24 hours, the cells were moved to liquid nitrogen for longer storage.

Use 1-2 vials of preclinical fetal joint cartilage, fetal tendon, or fetal skin to prepare a consistent working cell bank (WCB) for characterization. The design of the program is the same as when the cGMP was manufactured. Briefly, cells were initially placed at 1,500 cells / cm ² or 1,000-100,000 cells / cm ² in 100 cell culture flasks containing 15 ml of nutrient medium (DMEM + 10% clinical-grade fetal bovine serum, Invitrogen) ( T75, Nunc). The culture medium with cells was changed every 2 days, and the production / proliferation of the cells was performed at 37 ° C and 10% humidity for 10-14 days, or at a higher density for 3-6 days. On Days 12-14, a 10T75 batch of flask was subjected to TrypLE digestion to separate cells from the flask and placed in a centrifuge tube. Before centrifugation, an equal volume of nutrient medium was added to each centrifuge tube to buffer the effect of TrypLE. Cell pellets obtained from all flasks were resuspended in 200 ml frozen medium solution (DMEM, serum, DMSO, ratio 5: 4: 1), and aliquoted into 100 Nunc frozen vials (1.5 ml), dilution 10 x 10 ⁶ cells / ml.

Cells were frozen at -80 ° C and frozen in a Nalgene Cryo 1 ° C freezing container (Nalgene) to achieve a cooling and freezing curve speed of -1 ° C / min. After 24 hours, the cells were moved to the gas phase of liquid nitrogen. The WCB is in the second passage and is used for cell characterization. The cells used are in various passage numbers, but mainly between 2-8.

Characterization of fetal chondrocytes: identity, stability, consistency, genetic stability

Throughout the applicant's entire study, fetal chondrocytes made into a cell bank were compared with BM-MSC cell banks made in their laboratory under the same technical conditions. MSC is an allogenic cell, and is one of the main other cell types that have been proposed and used in pre-clinical and clinical experiments of cartilage regeneration in tissues (however, mesenchymal cells are non-tissue-specific and must be Controlled to become another organization type). Furthermore, the MSC cell bank is relatively unstable and must be used below the fourth generation. The same method can be applied to fetal tendon-derived progenitor cells and fetal skin-derived progenitor cells.

Characterization of fetal cartilage compared to BM-MSC

The fetal joint chondrocyte cell bank has been compared with BM-MSC cells, the surface labeling is qualitatively analyzed by FACS analysis, and the stromal deposition for functional tests is qualitative. Fetal chondrocytes derived from the epiphyseal tissue of the joint have been isolated, and the parental cell bank has been frozen.

Marks that have been compared in preliminary experiments include: CD105: Endoglin. Part of the TGFBeta receptor complex. The main criteria for MSC positive selection.

CD90: Thy-1. The main criteria for MSC positive selection.

CD44: PTPRC. White blood cells mark. Criteria for bmMSC negative selection.

CD73: NT5E. The main criteria for MSC positive selection.

CD166: ALCAM.

(See Figures 8 and 9)

The same method can also be applied to fetal tendon-derived progenitor cells and fetal skin-derived progenitor cells.

Biocompatibility: Biocompatibility between fetal cartilage and hydrogel and matrix

Biocompatibility of different matrices for medical use was initially tested. In the initial experiments, various compositions of hydrogels, collagen, and some biodegradable polymers were used. For hydrogels, cells were cultured in a gel that had been inserted into a preformed agar model. This is necessary to prevent cells from attaching to the test tube and to allow three-dimensional growth. The model is prepared by injecting 1ml of molten agarose (20% agar, low melting point agar) into a 1.5ml sterile conical microcentrifuge tube (eppendorf) through a pipette, and then filling the liquid in each microcentrifuge tube. The agar was inserted into a 0.5 ml sterile conical microcentrifuge tube and allowed to solidify, and then the 0.5 ml microcentrifuge tube was withdrawn, leaving a conical insert. After adding the gel and cells, 100 μl of the medium was added to the surface of each tube with a pipette, and the medium was changed twice a week. The cells were cultured in a 37 ° C incubator at 95% relative humidity and 10% carbon dioxide for 1 week, 2 weeks, and 4 weeks.

For the preparation of the matrix, the investigation preliminary experiments cell seeding density (10 ³ to 10 ⁴ cells cm ²⁾ and a growth period (1-28 days), and to determine the optimal conditions for the transfer of fetal cells. Fetal cells from the 3rd or 4th passage and BM-MSC (MSCs up to 4th passage due to subsequent stability) were placed in 10 ml medium (DMEM, containing 10% FBS), and seeded on the substrate. The matrix containing the cells was placed in a 37 ° C incubator and cultured at 95% relative humidity and 10% carbon dioxide. An additional 30 ml of medium was then added after 1 hour. Matrix was changed twice a week with nutrient medium. Biocompatibility is also measured by contact tests by culturing cells on a tissue culture plate that already has a hydrogel or matrix. Visual analysis of cell growth and migration at the interface of hydrogel and matrix. After 1 week, 2 weeks, and 4 weeks, the samples were stained with Giemsa stain and taken photos (Sony CyberShot DSC-S70, Zeiss macro lens, Zoom 6x, 3.3 million pixels).

[Biological Material Storage]

Domestic Deposit Information [Please note according to the order of deposit organization, date, and number]

1. Institute for Food Industry Development 2012/12/24 BCRC960459

2. Institute for Food Industry Development 2012/12/24 BCRC960460

3. Institute of Food Industry Development 2012/12/24 BCRC960461

Information on foreign deposits [Please note according to the order of the depositing country, institution, date, and number]

1.UK European Collection of Cell Cultures (ECACC) 2012/7/3 12070301

2.UK European Collection of Cell Cultures (ECACC) 2012/7/3 12070302

3.UK European Collection of Cell Cultures (ECACC) 2012/7/3 12070303

Claims (9)

An in vitro non-enzymatic method for isolating, expanding and developing fetal cells selected from the group consisting of fetal epiphyseal chondrocytes, fetal Achilles tendon cells, or fetal skin fibroblasts, comprising the following steps: a) using A fetal sample is selected from fetal ulnar cartilage containing fetal epiphyseal chondrocytes; fetal Achilles tendon containing fetal Achilles tendon cells; or fetal abdominal skin containing fetal skin fibroblasts; b) the fetal ulnar cartilage, fetal heel Tendons or fetal abdomen skin samples are micro-sectioned and dispersed within the engravings by physically attaching to the scribed surface with a scalpel; c) using the fetal epiphyseal chondrocytes, fetal Achilles tendon cells or fetuses in vitro The conditions of the proliferation of abdominal skin fibroblasts are to culture the fetal ulnar cartilage, fetal Achilles tendon or fetal abdominal skin samples; d) After 5-7 days of growth, select and isolate the earliest attached fetal epiphyseal chondrocyte cell population, The earliest attached fetal Achilles tendon cell population and the earliest attached fetal skin fibroblast population. For example, the in vitro non-enzymatic method of the scope of patent application, wherein the fetal ulnar cartilage sample is a fetal proximal ulna callus. A fetal epiphyseal chondrocyte (FEC) cell line, which is obtained by an in vitro non-enzymatic method such as item 1 of the patent application scope, is named FE002-Cart, and the deposit number is BCRC 960459. A fetal Achilles tendon cell line, which is obtained by an in vitro non-enzymatic method such as item 1 of the patent application scope, is named FE002-Ten, and is registered as BCRC 960461. A fetal skin fibroblast cell line, which is obtained by an in vitro non-enzymatic method such as item 1 of the patent application scope, is named FE002-SK2, and is registered as BCRC 960460. For example, the fetal epiphyseal chondrocyte (FEC) cell line of the third patent application scope is used as a therapeutic agent. For example, the fetal Achilles tendon cell cell line No. 4 in the scope of patent application is used as a therapeutic agent. For example, the fetal skin fibroblast cell line in the scope of the patent application No. 5 is used as a therapeutic agent. A method for preparing a parental cell bank (PCB) from fetal tissue in vitro. The fetal tissue is composed of fetal epiphyseal tissue, fetal Achilles tendon tissue, and fetal skin tissue. The method includes the following steps: a) using a fetal sample, which is Selected from fetal ulnar cartilage containing fetal epiphyseal chondrocytes; fetal Achilles tendon containing fetal Achilles tendon cells; or fetal abdominal skin containing fetal skin fibroblasts; b) the fetal ulnar cartilage, fetal Achilles tendon, or fetal abdominal skin The sample is micro-sectioned and dispersed in the engraving by being physically attached to the scribed surface of the scalpel; c) the fetal epiphyseal chondrocytes, fetal Achilles tendon cells, or fetal abdominal skin fibroblasts are dispersed in vitro The fetal ulnar cartilage, fetal Achilles tendon, or fetal abdominal skin samples were cultured under conditions of proliferation; d) After 5-7 days of growth, select and isolate the earliest fetal epiphyseal chondrocyte cell population and the earliest attached fetus. Achilles tendon cell cell population and the earliest attached fetal skin fibroblast cell population; e) freezing the earliest attached fetal epiphyseal chondrocytes that have been isolated Cell groups, the first group cells attached Achilles fetal cells, fetal and attaching the first skin fibroblasts cell bank for the group.