A xeno-free culture system for mesenchymal stem cells and uses thereof

Abstract

The application relates to the technical field of stem cells, in particular to a mesenchymal stem cell culture system without heterogeneity and application thereof. The mesenchymal stem cell culture system without heterogeneity comprises a basic culture medium and an additive component, wherein the basic culture medium is DMEM / F12, and the additive component comprises: recombinant human transferrin, recombinant human epidermal growth factor, recombinant human basic fibroblast growth factor, recombinant human insulin, recombinant human interferon gamma, hydrocortisone, vitamin A, vitamin D2, vitamin B, human blood albumin, CTS KnockOut SR XenoFree, glutamine and dextran. The mesenchymal stem cell culture system without heterogeneity provided by the application improves the proliferation efficiency of primary culture and in-vitro expansion culture of mesenchymal stem cells, is high in safety, good in stability and suitable for large-scale popularization and application.

Description

Technical Field

[0001] This application relates to the field of stem cell technology, specifically to a heterologous culture system for mesenchymal stem cells and its applications. Background Technology

[0002] As a major type of stem cell used in cell therapy, mesenchymal stem cells (MSCs) play a crucial role in organ repair and tissue regeneration, and have broad application prospects. In recent years, the clinical translational application of MSCs has developed rapidly. However, current stem cell culture technologies are insufficient to meet the growing clinical demand. In related technologies, stem cell culture systems typically include bovine serum, leading to problems such as foreign substance contamination, low culture efficiency, and unstable quality of the cultured stem cells.

[0003] Therefore, there is an urgent need to develop a component-safe, heterologous-free culture system for primary culture and in vitro expansion of mesenchymal stem cells, in order to improve the efficiency of stem cell culture, reduce the risk of animal-derived heterologous contamination, and improve the controllability of product quality, thereby ensuring the safe clinical application of stem cells. Summary of the Invention

[0004] This application addresses at least one of the problems of the related technology in the following aspects.

[0005] Therefore, the first aspect of this application provides a heterologous culture system for mesenchymal stem cells, comprising a basic culture system and additives.

[0006] The basal culture medium is DMEM / F12, and the added components include: recombinant human transferrin, recombinant human epidermal growth factor, recombinant human basic fibroblast growth factor, recombinant human insulin, recombinant human interferon-γ, hydrocortisone, vitamin A, vitamin D2, vitamin B, human serum albumin, CTS KnockOut SR XenoFree, glutamine, and dextran.

[0007] In some embodiments, the added ingredient comprises:

[0008] 4-6 mg / L Recombinant human transferrin, 10-40 µg / L Recombinant human epidermal growth factor, 10-40 µg / L Recombinant human basic fibroblast growth factor, 10-20 mg / L Recombinant human insulin, 5-20 mg / L Recombinant human interferon-gamma, 5-10 mg / L Hydrocortisone, 0.01-0.05 mg / L Vitamin A, 0.05-0.10 mg / L Vitamin D2, 0.02-0.06 mg / L Vitamin B, 0.5-2.5 (v / v)% Human serum albumin, 1-5 (v / v)% CTS KnockOut SR XenoFree, 60-100 mg / L Glutamine, and 2-10 mg / L Dextran 40.

[0009] In some embodiments, the added ingredient comprises:

[0010] 4-6 mg / L Recombinant human transferrin, 10-40 µg / L Recombinant human epidermal growth factor, 10-40 µg / L Recombinant human basic fibroblast growth factor, 10-20 mg / L Recombinant human insulin, 5-20 mg / L Recombinant human interferon-gamma, 5-10 mg / L hydrocortisone, 0.01-0.05 mg / L Vitamin A, 0.05-0.10 mg / L Vitamin D2, 0.02-0.06 mg / L Vitamin B, 2-10 (v / v)% 25% human serum albumin, 1-5 (v / v)% CTS KnockOut SR XenoFree, 60-100 mg / L glutamine, and 2-10 mg / L dextran 40.

[0011] In some embodiments, the added ingredient comprises:

[0012] 5 mg / L recombinant human transferrin, 35 µg / L recombinant human epidermal growth factor, 25 µg / L recombinant human basic fibroblast growth factor, 16 mg / L recombinant human insulin, 10 mg / L recombinant human interferon-gamma, 8 mg / L hydrocortisone, 0.03 mg / L vitamin A, 0.08 mg / L vitamin D2, 0.04 mg / L vitamin B, 1.25 (v / v)% human serum albumin, 5 (v / v)% CTS KnockOut SR XenoFree, 70 mg / L glutamine, and 3.5 mg / L dextran 40.

[0013] In some embodiments, the added ingredient comprises:

[0014] 5 mg / L recombinant human transferrin, 35 µg / L recombinant human epidermal growth factor, 25 µg / L recombinant human basic fibroblast growth factor, 16 mg / L recombinant human insulin, 10 mg / L recombinant human interferon-gamma, 8 mg / L hydrocortisone, 0.03 mg / L vitamin A, 0.08 mg / L vitamin D2, 0.04 mg / L vitamin B, 5 (v / v)% 25% human serum albumin, 5 (v / v)% CTS KnockOut SR XenoFree, 70 mg / L glutamine, and 3.5 mg / L dextran 40.

[0015] In some embodiments, the mesenchymal stem cells are derived from humans.

[0016] In some embodiments, the mesenchymal stem cells are selected from the group consisting of bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, placental mesenchymal stem cells, amniotic membrane mesenchymal stem cells, amniotic fluid mesenchymal stem cells, menstrual blood mesenchymal stem cells, peripheral blood mesenchymal stem cells, dental pulp mesenchymal stem cells, and skin mesenchymal stem cells.

[0017] In some embodiments, the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

[0018] The second aspect of this application provides the use of the heterologous culture system for mesenchymal stem cells described in any of the embodiments of the first aspect above in primary culture of mesenchymal stem cells or in vitro expansion of mesenchymal stem cells.

[0019] The embodiments of this application achieve the following beneficial effects:

[0020] The mesenchymal stem cell-free culture system provided in this application provides a method for primary culture or in vitro expansion culture of mesenchymal stem cells, which enables the stem cells to proliferate efficiently in vitro. The cultured stem cells have excellent morphology and high safety, while the culture cost is low, meeting the requirements for mass production of cell products and making them suitable for large-scale promotion and application. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The growth status of mesenchymal stem cells of various passages (P2-P10) obtained by amplification using the complete culture medium provided in this application in Example 1.

[0023] Figure 2 The viability of mesenchymal stem cells at each passage (P2-P10) obtained by amplification using the complete culture medium provided in this application in Example 1.

[0024] Figure 3 The unit area harvest density / cm² of mesenchymal stem cells from each passage (P2-P10) amplified using the complete culture medium provided in this application in Example 1. 2 .

[0025] Figure 4 The results show the proliferation capacity of P4 generation mesenchymal stem cells obtained by amplification using the complete culture medium provided in this application in Example 1.

[0026] Figure 5 The doubling time of mesenchymal stem cells amplified using the complete culture medium provided in this application in Example 1.

[0027] Figure 6 shows the differentiation capacity test results of P5 mesenchymal stem cells amplified using the complete culture medium provided in this application in Example 1. Figure 6A To differentiate into osteoblasts, Figure 6B To differentiate into adipocytes, Figure 6C To differentiate into chondrocytes.

[0028] Figure 7 The growth status of mesenchymal stem cells (P2-P10) obtained from the experimental and control groups in Comparative Example 1.

[0029] Figure 8 The figures show the mesenchymal stem cell proliferation curves of the experimental and control groups in Comparative Example 1.

[0030] Figure 9 The growth status of P2 to P6 generations of MSC-001 obtained by amplification using complete culture medium A (containing recombinant human interferon γ) and complete culture medium B (without recombinant human interferon γ) in Comparative Example 2 is shown.

[0031] Figure 10 To compare the number of cells harvested per bottle from passages P2 to P6 of MSC-001 obtained by amplifying complete culture medium A (containing recombinant human interferon γ) and complete culture medium B (without recombinant human interferon γ) in Comparative Example 2.

[0032] Figure 11 To compare the fold increases of P2 to P6 generations of MSC-001 obtained in Comparative Example 2, complete culture medium A (containing recombinant human interferon γ) and complete culture medium B (without recombinant human interferon γ) were used respectively. Detailed Implementation

[0033] The present invention will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the invention and are not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0034] This application is based on the inventor's following understanding:

[0035] In related technologies, mesenchymal stem cells are adult stem cells with self-replication ability and multi-directional differentiation potential. They are non-terminally differentiated cells and have characteristics of mesenchymal cells, endothelial cells, and epithelial cells.

[0036] Currently, mesenchymal stem cells (MSCs) can be isolated and identified from many human tissue sources, including fetuses and adults, such as bone marrow, blood, umbilical cord, cord blood, placenta, adipose tissue, amnion, amniotic fluid, dental pulp, and skin. Under specific in vitro induction conditions, MSCs can differentiate into various tissue cells, including adipose tissue, cartilage, bone, muscle, tendon, nerve, liver, myocardium, and endothelial cells. They retain their multi-lineage differentiation potential after continuous passage culture and cryopreservation. MSCs isolated from umbilical cord tissue exhibit stronger proliferative and differentiation capabilities, high in vitro proliferative potential, low immunogenicity, ease of isolation, high purity, and freedom from tumor cell contamination.

[0037] In recent years, the clinical translational application of mesenchymal stem cells (MSCs) has developed rapidly. As a major stem cell type widely used in cell therapy, MSCs play an important role in organ repair and tissue regeneration, and have broad application prospects. In the production of MSC therapy products, the culture medium used should meet the following quality standards: its components should have sufficient purity and be sterile, free of pathogenic microorganisms and endotoxins; residual culture medium should have no adverse effects on the subjects; and it should not affect the biological activity of stem cells, i.e., their "steminess" and differentiation capacity, while ensuring normal stem cell growth. The culture medium for culturing stem cells should also avoid using serum from animal and human sources as much as possible. Therefore, establishing a MSC culture system that is safe in composition, free of allogeneic components, has good cell adhesion, high proliferation efficiency, and is safe for clinical use is fundamental to improving the efficiency of in vitro stem cell culture and ensuring the safety of clinical applications, and is a necessary prerequisite for carrying out related MSC therapies.

[0038] In response, the inventors conducted extensive research and, by adding recombinant human transferrin, recombinant human epidermal growth factor, recombinant human basic fibroblast growth factor, recombinant human insulin, recombinant human interferon-γ, hydrocortisone, vitamin A, vitamin D2, vitamin B, human serum albumin, CTS KnockOut SR XenoFree, glutamine, and dextran to the basal culture medium DMEM / F12, obtained a safe and highly efficient heterologous culture system. This culture system can be used for the primary culture and in vitro expansion of human mesenchymal stem cells. The recombinant human interferon-γ contained therein is typically used for the directed induction of mesenchymal stem cell differentiation. The inventors of this application discovered that by combining it with other specific additives in the heterologous culture system for mesenchymal stem cells provided in the embodiments of this application within a specific content range, the proliferation efficiency and quality of mesenchymal stem cells in the basal culture medium DMEM / F12 can be significantly improved.

[0039] The first aspect of this application provides a heterologous culture system for mesenchymal stem cells, comprising a basal culture medium and additives.

[0040] The basal culture medium is DMEM / F12, and the added components include: recombinant human transferrin, recombinant human epidermal growth factor, recombinant human basic fibroblast growth factor, recombinant human insulin, recombinant human interferon-γ, hydrocortisone, vitamin A, vitamin D2, vitamin B, human serum albumin, CTS KnockOut SR XenoFree, glutamine, and dextran.

[0041] In some embodiments, the added components comprise: 4-6 mg / L (e.g., 4.2 mg / L, 4.6 mg / L, 4.8 mg / L, 5.0 mg / L, 5.2 mg / L, 5.4 mg / L, 5.6 mg / L, 5.8 mg / L) recombinant human transferrin, 10-40 µg / L (e.g., 15 µg / L, 20 µg / L, 25 µg / L, 30 µg / L, 35 µg / L) recombinant human epidermal growth factor (rhEGF), 10-40 µg / L (e.g., 15 µg / L, 20 µg / L, 25 µg / L, 30 µg / L, 35 µg / L) recombinant human basic fibroblast growth factor (rh-bFGF), 10-20 mg / L (e.g., 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L, 16 mg / L) Recombinant human insulin, 5-20 mg / L (e.g., 7.5 mg / L, 10 mg / L, 12.5 mg / L, 15 mg / L, 17.5 mg / L); Recombinant human interferon gamma, 5-10 mg / L (e.g., 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L); Hydrocortisone, 0.01-0.05 mg / L (e.g., 0.02 mg / L, 0.03 mg / L, 0.04 mg / L); Vitamin A, 0.05-0.10 mg / L (e.g., 0.06 mg / L, 0.07 mg / L, 0.08 mg / L, 0.09 mg / L); Vitamin D2, 0.02-0.06 mg / L (e.g., 0.03 mg / L, 0.04 mg / L, 0.05 mg / L). Vitamin B, 0.5-2.5 (v / v)% (e.g., 0.75(v / v)%, 1(v / v)%, 1.25(v / v)%, 1.5(v / v)%, 1.75(v / v)%, 2(v / v)%, 2.25(v / v)%); Human serum albumin, 1-5 (v / v)% (e.g., 2(v / v)%, 3(v / v)%, 4(v / v)%); CTS KnockOut SR XenoFree (brand: GIBCO), 60-100 mg / L (e.g., 65 mg / L, 70 mg / L, 75 mg / L, 80 mg / L, 85 mg / L, 90 mg / L, 95 mg / L); Glutamine, and 2-10 mg / L (e.g., 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L). Dextran 40.

[0042] In some embodiments, the added components comprise: 4-6 mg / L (e.g., 4.2 mg / L, 4.6 mg / L, 4.8 mg / L, 5.0 mg / L, 5.2 mg / L, 5.4 mg / L, 5.6 mg / L, 5.8 mg / L) recombinant human transferrin, 10-40 µg / L (e.g., 15 µg / L, 20 µg / L, 25 µg / L, 30 µg / L, 35 µg / L) recombinant human epidermal growth factor (rhEGF), 10-40 µg / L (e.g., 15 µg / L, 20 µg / L, 25 µg / L, 30 µg / L, 35 µg / L) recombinant human basic fibroblast growth factor (rh-bFGF), 10-20 mg / L (e.g., 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L, 16 mg / L) Recombinant human insulin, 5-20 mg / L (e.g., 7.5 mg / L, 10 mg / L, 12.5 mg / L, 15 mg / L, 17.5 mg / L); Recombinant human interferon gamma, 5-10 mg / L (e.g., 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L); Hydrocortisone, 0.01-0.05 mg / L (e.g., 0.02 mg / L, 0.03 mg / L, 0.04 mg / L); Vitamin A, 0.05-0.10 mg / L (e.g., 0.06 mg / L, 0.07 mg / L, 0.08 mg / L, 0.09 mg / L); Vitamin D2, 0.02-0.06 mg / L (e.g., 0.03 mg / L, 0.04 mg / L, 0.05 mg / L). Vitamin B, 2-10 (v / v)% (e.g., 2 (v / v)%, 3 (v / v)%, 4 (v / v)%, 5 (v / v)%, 6 (v / v)%, 7 (v / v)%, 8 (v / v)%, 9 (v / v)%); 25% human serum albumin, 1-5 (v / v)% (e.g., 2 (v / v)%, 3 (v / v)%, 4 (v / v)%); CTS KnockOut SR XenoFree (brand: GIBCO), 60-100 mg / L (e.g., 65 mg / L, 70 mg / L, 75 mg / L, 80 mg / L, 85 mg / L, 90 mg / L, 95 mg / L); glutamine, and 2-10 mg / L (e.g., 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L); dextran 40.

[0043] In some embodiments, the added components comprise: 5 mg / L recombinant human transferrin, 35 µg / L recombinant human epidermal growth factor, 25 µg / L recombinant human basic fibroblast growth factor, 16 mg / L recombinant human insulin, 10 mg / L recombinant human interferon-gamma, 8 mg / L hydrocortisone, 0.03 mg / L vitamin A, 0.08 mg / L vitamin D2, 0.04 mg / L vitamin B, 1.25 (v / v)% human serum albumin, 5 (v / v)% CTS KnockOut SR XenoFree, 70 mg / L glutamine, and 3.5 mg / L dextran 40.

[0044] In some embodiments, the added ingredients comprise: 5 mg / L recombinant human transferrin, 35 µg / L recombinant human epidermal growth factor, 25 µg / L recombinant human basic fibroblast growth factor, 16 mg / L recombinant human insulin, 10 mg / L recombinant human interferon-gamma, 8 mg / L hydrocortisone, 0.03 mg / L vitamin A, 0.08 mg / L vitamin D2, 0.04 mg / L vitamin B, 5 (v / v)% 25% human serum albumin, 5 (v / v)% CTS KnockOut SR XenoFree, 70 mg / L glutamine, and 3.5 mg / L dextran 40.

[0045] The mesenchymal stem cell culture system provided in this application embodiment is applicable to mesenchymal stem cells from various sources, such as bone marrow, blood, umbilical cord, cord blood, placenta, fat, amnion, amniotic fluid, dental pulp, skin, etc.

[0046] In some embodiments, the mesenchymal stem cells are derived from humans.

[0047] In some embodiments, the mesenchymal stem cells are selected from the group consisting of bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, placental mesenchymal stem cells, amniotic membrane mesenchymal stem cells, amniotic fluid mesenchymal stem cells, menstrual blood mesenchymal stem cells, peripheral blood mesenchymal stem cells, dental pulp mesenchymal stem cells, and skin mesenchymal stem cells.

[0048] In some embodiments, the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

[0049] The embodiments of the second aspect of this application provide the use of the heterologous culture system of mesenchymal stem cells of any of the embodiments of the first aspect above for primary culture of mesenchymal stem cells or in vitro expansion of mesenchymal stem cells.

[0050] In this application, the term "comprising" is an open-ended expression, meaning it includes the content specified in this invention, but does not exclude other aspects.

[0051] The following embodiments are used to further illustrate the advantages and features of this method, and are not intended to limit the invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in the field or according to the product instructions.

[0052] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Unless otherwise specified, all quantitative analysis experiments in the following examples were performed in triplicate, and the results were averaged.

[0053] Example 1: Primary isolation and in vitro expansion culture of human umbilical cord mesenchymal stem cells

[0054] 1. Establishment of a culture system for human umbilical cord mesenchymal stem cells

[0055] 1.1 Acquisition of the human umbilical cord

[0056] Eligible umbilical cord donors are primiparous or multiparous women aged 23-42 years, gestational age 30-40 weeks, with no pregnancy complications, no past medical history, no family medical history, and no infectious diseases.

[0057] On the day of delivery, a cesarean section was performed. After the baby was delivered, the umbilical cord was routinely ligated. A hemostat was used to clamp the cord 2-3 cm from the placental end, and then a surgical cord (umbilical cord loop) was tied around the inner end near the hemostat. After disinfection with a 0.5% iodine swab, the cord was cut between the hemostat and the ligation point. The same ligation was performed at the cut end of the cord more than 20 cm from the placental ligation point, ensuring the cord length between the two ligation points was greater than 20 cm. Both ends were then tied with sterile silk sutures. The cord was then preserved in umbilical cord preservation solution and transported to the laboratory.

[0058] 1.2 Preparation of human umbilical cord mesenchymal stem cell culture system (referred to as complete culture medium in this embodiment of the application)

[0059] (1) Select a 250 mL storage bottle as the container for the culture medium additive A, disinfect and sterilize the storage bottle, and then place it in a biosafety cabinet under aseptic conditions for later use.

[0060] (2) Using high-glucose DMEM / F12 as the basal culture medium, add the following to every 100 mL of basal culture medium: recombinant human transferrin: 5 mg, recombinant human epidermal growth factor (rhEGF): 35 µg, recombinant human basic fibroblast growth factor (rh-bFGF): 25 µg, recombinant human insulin: 16 mg, recombinant human interferon γ: 10 mg, hydrocortisone: 8 mg, vitamin A: 0.03 mg, vitamin D2: 0.08 mg, vitamin B: 0.04 mg, glutamine: 70 mg, and dextran 40: 3.5 mg. Mix the mixture by continuously pipetting to obtain crude additive A.

[0061] (3) The crude additive A prepared above is filtered through a 0.22 µm filter into a new storage bottle. The entire process is carried out under aseptic conditions until all the liquid is filtered. The liquid is dispensed into 10 mL / bottle and stored at -80℃ for later use as additive A.

[0062] (4) Prepare complete culture medium according to usage requirements: each 100 mL of basic culture medium contains 5 mL of 25% human serum albumin, 5 mL of CTS KnockOut SR XenoFree (GIBCO), 10 mL of additive A, and 80 mL of basic culture medium.

[0063] 1.3 Isolation and Culture of Primary Human Umbilical Cord Mesenchymal Stem Cells

[0064] You can choose any one of the following four methods, A, B, C, and D, as they have similar effects.

[0065] Method A: Cut the umbilical cord into several segments, remove the umbilical vein and artery, peel off Wharton's jelly with ophthalmic forceps, place it in a centrifuge tube, and cut it into approximately 1 mm pieces with surgical scissors. 3 Small pieces, per 1 mL (i.e., 1 cm) 3 Tissue blocks are seeded into one T75 culture flask or one 15 cm cell culture dish. A small amount of complete culture medium is used to disperse the tissue blocks into the flask or dish. After standing at room temperature for 2 hours, 15 mL of complete culture medium is added to each T75 culture flask or 15 cm cell culture dish. The flasks are then placed in a sterile incubator at 37°C, 5% CO2, and saturated humidity for incubation.

[0066] Method B: Cut the umbilical cord into several segments, remove the umbilical vein and umbilical artery, and place all remaining tissue in a centrifuge tube. Use surgical scissors to cut the tissue into pieces approximately 1 mm in size. 3Small pieces of tissue were seeded into one T75 culture flask or one 15 cm cell culture dish, with each 1 mL tissue piece seeded. The tissue pieces were dispersed into the flask or dish using a small amount of complete culture medium. After standing at room temperature for 2 hours, 15 mL of complete culture medium was added to each T75 culture flask or 15 cm cell culture dish. The flasks were then placed in a sterile incubator at 37°C, 5% CO2, and saturated humidity for incubation.

[0067] Method C: After cutting the umbilical cord into several segments and removing the umbilical vein and artery, Wharton's jelly is peeled off with ophthalmic forceps and placed in a centrifuge tube. Then, it is cut into pieces approximately 1 mm in size using surgical scissors. 3 Small tissue pieces were prepared by adding 1 mg / mL type I collagenase working solution (to tissue piece volume ratio approximately 3:1-5:1) and digesting at 37°C with shaking for 1-2 hours. Then, 0.1% trypsin working solution was added (to tissue piece volume ratio approximately 1:1-2:1) and digested at 37°C with shaking for 10-30 minutes. After digestion, DPBS was added to dilute the enzyme working solution in the reaction system. The cells were centrifuged at 500 g for 5 min, and the cell pellet was resuspended in complete culture medium. 4-6 mL tissue pieces were seeded into one T75 culture flask or one 15 cm cell culture dish. Complete culture medium was added to each T75 flask / cell culture dish, bringing the total complete culture medium volume to 15 mL. The cells were then incubated in a sterile incubator at 37°C, 5% CO2, and saturated humidity.

[0068] Method D: Cut the umbilical cord into several segments, remove the umbilical vein and artery, and place them directly into centrifuge tubes. Use surgical scissors to cut them into pieces approximately 1 mm in size. 3 Small tissue pieces were prepared by adding 1 mg / mL type I collagenase working solution (to tissue piece volume ratio approximately 3:1-5:1) and digesting at 37°C with shaking for 1-2 hours. Then, 0.1% trypsin working solution was added (to tissue piece volume ratio approximately 1:1-2:1) and digested at 37°C with shaking for 10-30 minutes. After digestion, DPBS was added to dilute the enzyme working solution in the reaction system. The cells were centrifuged at 500 g for 5 min, and the cell pellet was resuspended in complete culture medium. 4-6 mL tissue pieces were seeded into one T75 culture flask or one 15 cm cell culture dish. Complete culture medium was added to each T75 flask / cell culture dish, bringing the total complete culture medium volume to 15 mL. The cells were then incubated in a sterile incubator at 37°C, 5% CO2, and saturated humidity.

[0069] 1.4 In vitro expansion and culture of primary mesenchymal stem cells

[0070] (1) Mesenchymal stem cell passage can be performed when cell colonies are formed in the culture flask or culture dish and the cell fusion rate is about 0-90%.

[0071] (2) Discard the original culture supernatant, add DPBS and carefully wash once, then discard the washing solution.

[0072] (3) Add 2 mL of 0.1% trypsin to each T75 culture flask or each 15 cm cell culture dish, ensuring that the bottom of the flask or dish is moistened. Let it stand at room temperature for 3 minutes, then gently tap it and collect the digestion solution into a centrifuge tube. Carefully wash the tube once with DPBS, collect the washing solution into a centrifuge tube, and centrifuge at 500 g for 5 minutes.

[0073] (5) Discard the supernatant after centrifugation, resuspend the cell pellet in complete culture medium, adjust the cell density to a suitable level, and perform automatic or manual cell counting.

[0074] (6) 7000-12000 live cells / cm³ 2 The harvested cells were densely seeded into new culture flasks or dishes, supplemented with sufficient complete culture medium, and cultured in a sterile incubator at 37°C, 5% CO2, and saturated humidity.

[0075] 1.5 Cryopreservation of Mesenchymal Stem Cells

[0076] (1) Mesenchymal stem cells can be cryopreserved when the cell confluence in the culture flask or culture dish is about 60-90%.

[0077] (2) Discard the original culture supernatant, add DPBS and carefully wash once, then discard the washing solution.

[0078] (3) Add 2 mL of 0.1% trypsin to each T75 culture flask or each 15 cm cell culture dish, ensuring that the bottom of the flask or dish is moistened. Let it stand at room temperature for 3 minutes, then gently tap it and collect the digestion solution into a centrifuge tube. Carefully wash the tube once with DPBS, collect the washing solution into a centrifuge tube, and centrifuge at 500 g for 5 minutes.

[0079] (4) Discard the supernatant after centrifugation, resuspend the cell pellet in commercial cryopreservation solution (brand: GIBCO), mix well by pipetting, and then dispense into cryovials.

[0080] (5) Place the cryovials in a programmed cooling box (brand: Corning) and store at -80℃ for 12-72 hours, then transfer them into liquid nitrogen for long-term storage.

[0081] 2. Quality evaluation of mesenchymal stem cells cultured using a heterologous clinical-grade mesenchymal stem cell culture system (i.e., the complete culture medium provided in this embodiment).

[0082] 2.1 Cellular characteristics

[0083] 2.1.1 Cell morphology:

[0084] It exhibits typical elongated spindle-shaped, swirling growth, such as Figure 1 As shown.

[0085] 2.12 Cell viability:

[0086] At each harvest, the cell viability generally remains above 90%, such as Figure 2 The survival rates of generations P2 to P10 are 88.94%, 89.30%, 91.23%, 93.21%, 94.06%, 93.97%, 91.68%, 90.55%, and 90.01%, respectively.

[0087] 2.13 Cell yield per unit area:

[0088] The cell harvest density per unit area per generation is maintained at 4 × 10⁻⁶. 4 Up to 9.5×10 4 / cm 2 Left and right, such as Figure 3 As shown.

[0089] 2.14 Cell phenotype and purity:

[0090] Mesenchymal stem cells at passages P5 and P10 were analyzed by flow cytometry. The positive rates of CD73, CD90, and CD105 were all ≥95%, while the positive rates of CD133, CD11b, CD19, CD31, CD34, CD45, CD33, HLA-DR, and CD14 were all ≤2%, as shown in Tables 1 and 2.

[0091] Table 1. Results of P5 generation cell phenotype and purity detection

[0092]

[0093] Table 2. Results of P10 generation cell phenotype and purity detection

[0094]

[0095] 2.15 Cell proliferation capacity:

[0096] The proliferation capacity of P4 generation mesenchymal stem cells was detected using CCK8 assay; the proliferation curve showed a standard "S" shape. Figure 4 As shown.

[0097] 2.16 Cell Cycle:

[0098] The proportion of cells in the S and G2 phases of a specific passage mesenchymal stem cell population was detected using a cell cycle assay kit (brand: Beyotime). The proportion of cells in the S+G2 phase was approximately 20-40%, as shown in Table 3.

[0099] Table 3. Results of cell cycle analysis at P4 passage of mesenchymal stem cells

[0100]

[0101] 2.17 Cell doubling time:

[0102] Calculate the doubling time using the formula: Doubling time = t lg2 / (lgNt-lgN0), where Nt refers to the number of harvested cells and N0 refers to the number of inoculated cells. The calculation results are as follows. Figure 5 As shown, the doubling times for passages P2 to P10 were 24.75 h, 21.46 h, 22.04 h, 21.79 h, 24.71 h, 23.48 h, 28.10 h, 29.33 h, and 31.23 h, respectively. Among these, passages P2-P5 showed good cell proliferation with doubling times within 22 h; passages P6-P9 showed doubling times within 30 h, with a slower overall cell proliferation rate but a steady and relatively stable increase in the total cell count; as the cell passages increased, cells gradually aged, and the doubling time for passage P10 exceeded 30 h. The cell doubling time gradually increased with each passage, as the proliferation rate slowed down. Figure 5 As shown.

[0103] 2.18 The results of cell growth support assays are shown in Table 4 below. The expansion fold = number of harvested cells / inoculation area × inoculation density, and the inoculation area for each passage was 150 cm². 2 Furthermore, the total number of harvested cells is calculated based on the total expansion factor after increasing the inoculation area by 20 times. For example, the total number of harvested cells for P2 is 8000 cells / cm². 2 ×150 cm 2 ×20×195=468×10 7 .

[0104] Table 4 Cell growth support test

[0105]

[0106] 2.19 Multidirectional Differentiation:

[0107] Osteoblastic, adipogenic, and chondrogenic differentiation induction kits (all from Cyagen Biosciences) were used to induce osteogenic, adipogenic, and chondrogenic differentiation of specific passage (P5) mesenchymal stem cells. All mesenchymal stem cells could differentiate into osteoblasts (…). Figure 6A ), fat cells ( Figure 6B ) and cartilage globules ( Figure 6C Differentiation, as shown in Figure 6.

[0108] Example 2. Quality evaluation and standards of the allogeneic clinical-grade human mesenchymal stem cell culture system (i.e., the complete culture medium provided in Example 1 of this application).

[0109] 1. Quality evaluation and standards for additive A

[0110] The following tests were conducted according to the 2020 edition of the Pharmacopoeia of the People's Republic of China:

[0111] 1.1 Appearance and Visible Foreign Objects

[0112] The quality control test result was a colorless, clear, and transparent liquid, which meets the quality control standards, and the result is judged to be qualified.

[0113] 1.2 Osmotic pressure

[0114] The quality control test result was 315 mOsm / kg, which is within the quality control standard range of 280-350 mOsm / kg, and the result was deemed qualified.

[0115] 1.3 pH value:

[0116] The quality control test result was 7.30, which is within the quality control standard range of 6.8-7.8, and the result was judged to be qualified.

[0117] 1.4 Endotoxin detection:

[0118] The quality control result is <0.25 EU / mL, which meets the quality control standard, and the result is judged to be qualified.

[0119] 1.5 Sterility test:

[0120] The quality control result was negative, which meets the quality control standards, and the result is judged to be qualified.

[0121] 1.6 Mycoplasma detection:

[0122] The quality control result was negative, which meets the quality control standards, and the result is judged to be qualified.

[0123] 2. Quality evaluation and standards for complete mesenchymal stem cell culture medium

[0124] 2.1 Appearance and Visible Foreign Objects:

[0125] The quality control test result was a pale yellow, clear, and transparent liquid, which met the quality control standards, and the result was deemed qualified.

[0126] 2.2 Osmotic pressure:

[0127] The quality control test result was 312 mOsm / kg, which is within the quality control standard range of 280-350 mOsm / kg, and the result was deemed qualified.

[0128] 2.3 pH value:

[0129] The quality control test result was 7.35, which is within the quality control standard range of 6.8-7.8, and the result was judged to be qualified.

[0130] 2.4 Endotoxin detection:

[0131] The quality control result is <0.25 EU / mL, which meets the quality control standard, and the result is judged to be qualified.

[0132] 2.5 Sterility test:

[0133] The quality control result was negative, which meets the quality control standards, and the result is judged to be qualified.

[0134] 2.6 Mycoplasma detection:

[0135] The quality control result was negative, which meets the quality control standards, and the result is judged to be qualified.

[0136] 2.7 Quality control standards for control cells

[0137]

[0138] Comparative Example 1.

[0139] 1.1 Cell adhesion effect

[0140] Human umbilical cord mesenchymal stem cells from passages MSC-001 P5, MSC-002 P5, and MSC-003 P5 were collected. Three parallel experimental groups and three parallel control groups were set up for each batch. 1.5 × 10⁻⁶ cells were used per well. 5 Cells were seeded at a rate of 100 cells / mL in 24-well plates. The control group was prepared with basal medium (high glucose DMEM / F12), and the experimental group was prepared with complete medium as described in Example 1. After seeding, the cells were incubated at 37°C in a 5% CO2 cell culture incubator for 2 h. The cells and their adhesion were observed under an inverted microscope. The cell culture supernatant was discarded, and the cells were washed with DPBS, digested with TryPLE, and counted. The results are shown in Table 5 below.

[0141] Table 5. Number of mesenchymal stem cells (MSC-001 P5, MSC-002 P5, MSC-003 P5) adhering to the culture vessel (cells)

[0142]

[0143] The results show that, as can be seen from the table above, the number of cells adhering to the experimental group was significantly higher than that of the control group. The control group's culture medium contained only basal medium without any other substances, and its cell adhesion performance was worse than that of the experimental group. In the inter-group comparison of the three batches of cells, the number of cells adhering to the experimental group was higher than that of the control group. In the three parallel controls within the group, the number of cells adhering to the experimental group was also higher than that of the control group. This indicates that the complete culture medium provided in the embodiments of this application can significantly improve the cell adhesion performance.

[0144] 1.2 Cell growth status

[0145] Human umbilical cord mesenchymal stem cells (MSC-001 P1 passage) were harvested and resuscitated, with a cell quantity of 1×10⁻⁶. 6 Cells / branch, determined by cell counting at 8000 cells / cm². 2 Cells were seeded at different densities into two T75 cell culture flasks. The experimental and control groups were set up in the same manner as in comparative 1.1. After being shaken well, the cells were placed in a 37°C, 5% CO2 incubator for culture.

[0146] Cell passage: When the cell confluence reaches 80-90%, perform passage culture. First, discard the cell culture supernatant, wash the cells twice with an appropriate amount of DPBS, add TryPLE to digest at room temperature for 2-3 minutes. After the cells detach, add an appropriate amount of DPBS to reduce the digestion effect, and pipette 3-4 times to make the cells into a single cell state. Transfer the cell suspension to a centrifuge tube; then take an appropriate amount of DPBS to wash the bottom of the flask, and transfer it to the same centrifuge tube. Centrifuge at 500g for 5 minutes.

[0147] Cell counting: After centrifugation, discard the supernatant, add an appropriate amount of culture medium to the centrifuge tube and pipette 3-4 times to resuspend and mix the cells. Count the cells and seed them into T75 culture flasks at a density of approximately 8000 cells / cm². Shake well and incubate in a 37°C, 5% CO2 incubator.

[0148] Cell morphology was observed and recorded under an inverted microscope during continuous culture up to the 10th generation (e.g., ...). Figure 7 As shown in the figure, the cultured cells were digested with TryPLE enzyme solution at each passage, and the cell count was performed using a cell counting chamber to calculate the cell viability. A cell density proliferation curve was plotted as a function of culture time, and the results are shown in the figure. Figure 8 As shown.

[0149] Table 6 Summary of doubling rates and doubling times for different generations

[0150]

[0151] The results showed that, as can be seen from the cell growth status diagram, during 10 consecutive days of cell culture, the cells cultured in the complete culture medium provided in Example 1 of this application (corresponding to the experimental group) had a higher number of adherent cells than those cultured in the basal culture medium (corresponding to the control group). The cells were in good growth condition, neatly arranged, and had a relatively plump morphology. In contrast, the proliferation rate of the control group was slower than that of the experimental group. This indicates that the complete culture medium provided in Example 1 of this application improves the adhesion effect of stem cells and can also increase the proliferation rate of stem cells. As can be seen from Table 6 above, the cell doubling rate was the highest at passage P3, and the cell doubling rate remained stable from passage P4 to P8. From passage P9 to P10, the cells may show a senescence trend and the proliferation capacity slowed down. The cell proliferation rate achieved by culturing stem cells in the complete culture medium with added supplementary components provided in this application can meet the current needs of cell production experiments.

[0152] 1.3 CCK-8 assay for cell population doubling

[0153] Human umbilical cord mesenchymal stem cells (MSC-001 P1 passage) were harvested and resuscitated, with a cell quantity of 1×10⁻⁶. 6 Cells / branch, determined by cell counting at 8000 cells / cm². 2 Cells were seeded into T25 cell culture flasks at different densities. The experimental and control groups were set up in the same manner as the control group 1.1. After shaking well, the cells were placed in a 37°C, 5% CO2 incubator for culture. When the cell confluence reached 80-90%, the cells were harvested and counted.

[0154] Adjust the remaining cell density to 1×10 4 Cells / mL: 100 µL of cell suspension was added to one well of a 96-well cell culture plate, with five replicates per sample. A set of wells containing only complete mesenchymal culture medium was added to each plate as a blank control, also with five replicates. A total of eight 96-well plates were seeded and labeled D1, D2, D3, D4, D5, D6, D7, and D8. After incubation at 37°C, 5% CO2 for 24 / 48 / 72 / 96 / 120 / 144 / 168 / 192 hours, one well of each plate was removed for analysis. To prevent errors caused by liquid evaporation, the wells at the edge of the 96-well plate were not used for experiments; only 200 μL of PBS solution was added.

[0155] After plating, one 96-well plate was taken out every 24 hours for OD value measurement: 10 µL of CCK8 solution was added to each well of both the experimental and control groups under dark conditions (Note: avoid air bubbles in the wells to prevent affecting the OD value), and the plate was incubated in an incubator for 3-4 hours. The incubation time must be the same for each experiment. The OD value was measured at 450 nm using a microplate reader. If the OD value is not measured immediately, 10 µL of 0.1 M HCl solution or 1% w / v SDS solution can be added to each well, and the plate can be covered and stored at room temperature in the dark; the absorbance will not change within 24 hours.

[0156] Cell population doubling time calculation:

[0157] Of the five replicates, the highest and lowest OD values ​​were removed, and the average of the remaining three replicates was taken. The average OD value of the sample cells minus the average OD value of the blank wells is the OD value of the cells tested on the day of the experiment. A growth curve was plotted with culture time on the x-axis and OD value on the y-axis, and the calculation was performed at the points on the linear portion of the logarithmic growth phase. The time used for data calculation should be the same for each group in each experiment.

[0158] The doubling time is calculated using the formula: PDT = t [lg2 / (lgNt2-lgNt1)], where t represents the logarithmic proliferation period (t2-t1) in the growth curve, Nt1 is the absorbance value measured 24 hours after inoculation, and Nt2 is the absorbance value after time t. Usually, t is taken as the logarithmic proliferation period, and Nt2 is taken as the end of the logarithmic proliferation period.

[0159] from Figure 8 As can be seen, under continuous culture in basal medium (control group) and complete medium (experimental group), the OD value of umbilical cord mesenchymal stem cells at 450 nm showed a continuous upward trend with increasing culture time. Overall, the proliferation rate of cells cultured in complete medium was consistently higher than that of cells cultured in the control medium. On day 7, the proliferation rate of cells cultured in the control medium reached an inflection point, and the proliferation trend slightly decreased. The OD value of cells cultured in complete medium showed little change on days 7 and 8, and the proliferation rate did not change significantly. This indicates that cells cultured in complete medium had better proliferation activity and better proliferation effect, suggesting that the additive components in the complete medium can promote cell proliferation ability, and the proliferation effect is significant compared to the control medium group.

[0160] Comparative Example 2

[0161] The culture medium provided in this comparative example differs from the complete culture medium provided in Example 1 only in that it does not contain recombinant human interferon γ.

[0162] 1. Preparation of complete culture medium for human umbilical cord mesenchymal stem cells

[0163] (1) Select a 250mL storage bottle as the container for the culture medium additives A and B, disinfect and sterilize the storage bottle, and then place it in a biosafety cabinet under aseptic conditions for later use.

[0164] (2) Prepare additives containing recombinant human interferon γ and those without recombinant human interferon γ respectively.

[0165] Additive A containing recombinant human interferon-gamma

[0166] Using high-glucose DMEM / F12 as the basal culture medium, the following additives were added to every 100 mL of basal culture medium: recombinant human transferrin: 5 mg, recombinant human epidermal growth factor (rhEGF): 35 µg, recombinant human basic fibroblast growth factor (rh-bFGF): 25 µg, recombinant human insulin: 16 mg, recombinant human interferon γ: 10 mg, hydrocortisone: 8 mg, vitamin A: 0.03 mg, vitamin D2: 0.08 mg, vitamin B: 0.04 mg, glutamine: 70 mg, and dextran 40: 23.5 mg. The mixture was continuously pipetted and stirred to obtain crude additive A.

[0167] Additive B without recombinant human interferon-gamma

[0168] Using high-glucose DMEM / F12 as the basal culture medium, add the following to every 100 mL of basal culture medium: recombinant human transferrin 5 mg, recombinant human epidermal growth factor (rhEGF) 35 µg, recombinant human basic fibroblast growth factor (rh-bFGF) 25 µg, recombinant human insulin 16 mg, hydrocortisone 8 mg, vitamin A 0.03 mg, vitamin D2 0.08 mg, vitamin B 0.04 mg, glutamine 70 mg, and dextran 4 5 mg. Mix thoroughly by continuous pipetting to obtain crude additive B.

[0169] (3) The above-mentioned crudely treated additives A and B are filtered through a 0.22 µm filter into a new storage bottle. The entire process is carried out under aseptic conditions until all the liquid is filtered. The liquid is dispensed into 10 mL / bottle and stored at -80℃ to obtain additives A and B for later use.

[0170] (4) Prepare complete culture media A and B according to the usage requirements: each 100 mL of basic culture medium contains 5 mL of 25% human serum albumin, 5 mL of CTS KnockOut SR XenoFree (GIBCO), 10 mL of additive A or B, and 80 mL of basic culture medium.

[0171] 2. Resuscitation and passage of MSC-001 P1 generation mesenchymal stem cells

[0172] Human umbilical cord mesenchymal stem cells (MSC-001 P1 passage) were harvested and resuscitated, with a cell quantity of 1×10⁻⁶. 6 Cells / branch, determined by cell counting at 8000 cells / cm². 2 Cells were seeded into two T75 cell culture flasks at different densities, using complete medium A containing recombinant human interferon γ and complete medium B without recombinant human interferon γ, respectively. After shaking well, the flasks were placed in a 37°C, 5% CO2 incubator for culture.

[0173] Cell passage: When the cell confluence reaches 80-90%, proceed with passage culture. First, discard the cell culture supernatant, wash the cells twice with an appropriate amount of DPBS, add TryPLE to digest at room temperature for 2-3 minutes. After the cells detach, add an appropriate amount of DPBS to reduce the digestion effect, and pipette 3-4 times to make the cells into a single cell state. Transfer the cell suspension to a centrifuge tube; then take an appropriate amount of DPBS to wash the bottom of the flask, and transfer it to the same centrifuge tube. Centrifuge at 500g for 5 minutes.

[0174] Cell counting: After centrifugation, discard the supernatant, add an appropriate amount of culture medium to the centrifuge tube and pipette 3-4 times to resuspend and mix the cells. Count the cells and seed them into T75 culture flasks at a density of approximately 8000 cells / cm². Shake well and incubate at 37°C in a 5% CO2 incubator.

[0175] During the continuous culture up to the 6th generation, cell morphology was observed and recorded under an inverted microscope. At each passage, the cultured cells were digested with TryPLE enzyme solution, and cell counts were performed using a cell counting chamber to calculate cell viability and fold increase. A graph showing the change in the number of cells harvested per flask at each passage generation was plotted, along with the cell morphology results for each passage. Figure 9 As shown.

[0176] from Figure 9 As can be seen, MSC-001 P1 passage cells were revived and cultured to P6 passages in complete medium A and complete medium B, respectively. Under the same conditions, the cells cultured in complete medium A had higher overall confluence and adherence than those cultured in complete medium B, and there were relatively fewer dead cells floating in the culture supernatant. Cells cultured in complete medium B at passage P6 were flat, narrow, and sparse, and their cell proliferation ability was relatively poor.

[0177] Cell counts were performed on P2-P6 passages cultured in complete culture medium A and complete culture medium B. The number of cells harvested per flask for both types of complete culture media is as follows: Figure 10 As shown, Figure 10The results clearly show that the number of cells harvested per flask from complete culture medium A was higher than that from complete culture medium B at all cell passages, while the number of cells harvested per flask from complete culture medium B did not exceed 3 × 10⁻⁶. 6 The highest number of cells harvested from a single flask in complete culture medium A can reach 7 × 10⁶. 6 Approximately 1, with a minimum of about 4×10. 6 The number of cells harvested per flask at each passage using complete culture medium A was higher than that using complete culture medium B.

[0178] The fold increase of cells cultured in complete media A and B was calculated, starting from... Figure 11 As can be seen from the diagram, the blue line represents the fold increase of each passage in the complete culture medium A group, while the red line represents the fold increase of each passage in the complete culture medium B group. The cells cultured using complete culture medium A can achieve a fold increase of up to 3.5, while the cells cultured using complete culture medium B can achieve a fold increase of up to 2. The trend of fold increase across each passage is higher in the blue line than in the red line, indicating that cells cultured using complete culture medium A are superior to cells cultured using complete culture medium B.

[0179] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0180] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A heterologous-free culture system for mesenchymal stem cells, comprising a basal culture medium and additives. The basal culture medium is high-glucose DMEM / F12, and the added components consist of the following: 4-6 mg / L recombinant human transferrin 10-40 µg / L recombinant human epidermal growth factor 10-40 µg / L recombinant human basic fibroblast growth factor 10-20 mg / L recombinant human insulin 5-20 mg / L recombinant human interferon-gamma 5-10 mg / L hydrocortisone 0.01-0.05 mg / L Vitamin A 0.05-0.10 mg / L Vitamin D2 0.02-0.06 mg / L Vitamin B, 2-10 (v / v)% 25% human serum albumin 1-5 (v / v)% CTS KnockOut SR XenoFree, 60-100 mg / L glutamine, and 3.5 mg / L dextran 40.

2. The use of the heterologous culture system for mesenchymal stem cells as described in claim 1 in primary culture of umbilical cord mesenchymal stem cells or in vitro expansion of umbilical cord mesenchymal stem cells.

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