WO2017096619A1

WO2017096619A1 - Reagent kit used for cell culture

Info

Publication number: WO2017096619A1
Application number: PCT/CN2015/097156
Authority: WO
Inventors: 郭镭
Original assignee: 郭镭; 里程
Priority date: 2015-12-11
Filing date: 2015-12-11
Publication date: 2017-06-15

Abstract

Provided is a novel reagent kit for serum-free stem cell culture, the reagent kit comprising a serum-free culture medium, the ingredients of said culture medium comprising: 0.05-2 parts by volume of β-mercaptoethanol, 0.5-2 parts by volume of non-essential amino acid aqueous solution, 4-6 parts by volume of human mesenchymal stem cell culture supernatant concentrate, and 90-95 parts by volume of a-MEM/DMEM-F12 and recombinant human alkaline fibroblast growth factor of a final concentration of 5-5 ng/ml.

Description

A kit for culturing cells

Technical field

The invention relates to the research field of stem cells, in particular to a novel and high-efficiency serum-free medium, a preparation method thereof and use thereof.

Background technique

Mesenchymal stem cells are ubiquitous in various tissues and organs of human body, and have multi-directional differentiation potential. They have functions of stimulating tissue regeneration and regulating immunity, and have broad application prospects in the field of cell therapy.

Bone marrow mesenchymal stem cells have been widely used clinically, and current studies have shown that umbilical cord-derived mesenchymal stem cells can not only be an ideal substitute for bone marrow mesenchymal stem cells, but also have greater application potential. Among them, human Umbilical Cord mesenchymal stem cells (hUC-MSCs) express unique markers of various embryonic stem cells, which have large differentiation potential, strong proliferation ability, low immunogenicity and convenient materials. , no restrictions on ethical issues, easy to industrialize and other characteristics, and studies have confirmed that hUC-MSCs have good therapeutic effects in animal models and clinical studies of diseases such as neurological diseases, immune system, endocrine system, cancer, heart disease, etc. It may become the most versatile stem cell for clinical application.

In order to further apply hUC-MSCs to the clinic, the most important thing is that the large-scale expansion of hUC-MSCs in vitro reaches an effective clinical therapeutic dose. Therefore, in vitro culture of hUC-MSCs has become one of the most basic and most important technologies. The existing hUC-MSCs culture methods mostly use FBS and streptomycin in the basal medium, but the non-human serum components are complex, which makes hUC-MSCs easy to differentiate in the long-term culture process, and there is a danger of spreading heterologous pathogens. .

In addition, although researchers have developed various types of serum substitutes, the market-purchased serum substitutes for hUC-MSCs culture and complete medium culture are still not ideal, especially for stem cell attachment. Characteristics such as proliferation, proliferation, and maintenance of stability after long-term cell culture did not achieve the desired results.

Summary of the invention

In view of the above technical problems, the inventors of the present invention have found that during the culture of hUC-MSCs, the cells secrete a variety of factors, proteins and the like which are beneficial to their own growth, and can be used to perform cells in a serum-free environment. In particular, long-term expansion culture of stem cells.

It is therefore an object of the present invention to provide a kit for culturing cells, the kit comprising a serum-free medium containing a concentrate obtained from a cell culture, particularly suitable in a serum-free environment Cultured cells.

The technical solution of the present invention is as follows.

In one aspect, the invention provides a kit for culturing cells, the kit comprising a serum-free medium comprising a-MEM/DMEM-F12, β-mercaptoethanol, non-essential amino acids , a culture supernatant of recombinant human basic fibroblast growth factor (b-FGF) and human umbilical cord mesenchymal stem cells (hUC-MSCs).

Preferably, the serum-free medium comprises 0.05-0.2 parts by volume of β-mercaptoethanol, 0.5-2 parts by volume of a non-essential amino acid aqueous solution, 4-6 parts by volume of a culture supernatant concentrate, and 90-95 parts by volume. a-MEM/DMEM-F12 and recombinant human basic fibroblast growth factor at a final concentration of 5-15 ng/ml, wherein the non-essential amino acid aqueous solution comprises glycine, alanine, L- at a concentration of 8-12 mM each. Tianmenamide, L-aspartic acid, glutamic acid, proline and serine;

More preferably, the serum-free medium comprises 0.1 parts by volume of β-mercaptoethanol, 1 part by volume of an aqueous solution of non-essential amino acids, 5 parts by volume of a culture supernatant, and 94 parts by volume of a-MEM/DMEM-F12. And recombinant human basic fibroblast growth factor at a final concentration of 10 ng/ml.

The culture supernatant concentrate is obtained by a method comprising the following steps:

Human umbilical cord mesenchymal stem cell culture supernatant was collected, centrifuged, filtered through a microfiltration membrane, and concentrated by ultrafiltration.

Preferably, the culture supernatant concentrate is obtained by a method comprising the following steps:

(1) Human umbilical cord mesenchymal stem cells are seeded in serum-free medium at a density of 0.5-4×10 ⁴ cells/cm ² for 48-72 hours to allow cells to reach 70%-90% confluence, and fresh umbilical cords are collected. Mesenchymal stem cell culture supernatant;

(2) Centrifuge at 3000-5000 g for 15-40 min at 4 ° C to remove cells and cell debris suspended in the culture supernatant;

(3) The supernatant obtained in the step (2) is centrifuged at 10000 g for 30-60 min at 4 ° C to remove cytoplasm and other impurities in the supernatant;

(4) The supernatant obtained in the step (3) is sterilized by a 0.22 μm microfiltration membrane;

(5) The filtrate obtained in the step (4) is transferred into a 3KD ultrafiltration concentrating tube, centrifuged at 3000-4500 g for 60-90 min at 4 ° C, and the filtrate is concentrated to 1/20 to 1 of its volume. /50;

(6) The concentrate obtained in the step (5) was adjusted to a final volume of PBS buffer to be 1/20 of the cell culture supernatant collected in the step (1).

In the step (1), the human umbilical cord mesenchymal stem cells (hUC-MSCs) are preferably human umbilical cord mesenchymal stem cells isolated from fresh umbilical cord tissue of a healthy newborn born or cesarean section.

And preferably, the serum-free medium in step (1) contains a-MEM/DMEM-F12, β-mercaptoethanol, non-essential amino acids, recombinant human basic fibroblast growth factor (b-FGF), and optionally human a culture supernatant concentrate of umbilical cord mesenchymal stem cells; preferably comprising 0.05-0.2 parts by volume of β-mercaptoethanol, 0.5-2 parts by volume of a non-essential amino acid aqueous solution, optionally 4-6 parts by volume of a culture supernatant concentrate 90-95 parts by volume of a-MEM/DMEM-F12 and recombinant human basic fibroblast growth factor at a final concentration of 5-15 ng/ml, wherein the non-essential amino acid aqueous solution comprises glycine at a concentration of 8-12 mM each , alanine, L-aspartamide, L-aspartic acid, glutamic acid, valine, and serine; more preferably, 0.1 part by volume of β-mercaptoethanol, 1 part by volume of an aqueous solution of a non-essential amino acid, optionally 5 parts by volume of the culture supernatant concentrate, 94 parts by volume of a-MEM/DMEM-F12, and recombinant human basic fibroblast growth factor at a final concentration of 10 ng/ml.

The serum-free medium is prepared by a preparation method comprising the following steps:

The human umbilical cord mesenchymal stem cell culture supernatant is collected, centrifuged, filtered through a microfiltration membrane, and concentrated by ultrafiltration to obtain a culture supernatant concentrate;

Preparing a premix with β-mercaptoethanol, non-essential amino acids, and a-MEM/DMEM-F12;

The culture supernatant, the premix, and the recombinant human basic fibroblast growth factor are mixed.

Wherein, the culture supernatant concentrate is preferably obtained by a method comprising the following steps:

(2) Centrifuge at 3000-4500g for 15-40 minutes at 4 ° C to remove the suspension in the culture supernatant. Cells and cell debris;

The kits provided by the present invention are useful for culturing cells, particularly stem cells, and thus also for the use of the kits in stem cell culture, preferably serum-free culture. Wherein the stem cells of the present invention are isolated from a mammal, such as a human tissue or organ, the tissue or organ being one or more of bone marrow, umbilical cord and fat; preferably, the stem cell is a mammalian source The umbilical cord mesenchymal stem cells are more preferably human-derived umbilical cord mesenchymal stem cells; further preferably, the stem cells are human umbilical cord mesenchymal stem cells isolated from fresh umbilical cord tissue of a healthy newborn born or cesarean section.

The inventors have found that human umbilical cord mesenchymal stem cells secrete a variety of factors, proteins and other components that are beneficial to the growth of the cells during the culture process. Based on this, the present invention provides a novel serum-free medium by using a concentrate obtained by concentrating human umbilical cord mesenchymal stem cell culture supernatant. The medium of the invention can be used for long-term expansion culture of stem cells in a serum-free environment, and the stem cells still maintain pluripotency and strong proliferation ability, and the stem cells can be bone marrow and umbilical cord from mammals (for example, humans). And one or more of the fats, especially the umbilical cord mesenchymal stem cells isolated from the umbilical cord of the newborn.

Specifically, the novel serum-free medium in the kit provided by the present invention does not contain a heterologous serum component, thereby avoiding the instability of the cell growth process due to serum batch differences during cell culture, and also precluding the transmission of xenogenic pathogens. The possibility of danger; and the use of the serum-free medium solves the shortcomings of poor cell adherence and slow proliferation in conventional serum-free culture, and maintains good proliferative capacity and multi-directional differentiation potential during long-term culture. It provides an efficient solution for in vitro culture of animal cells; in addition, the culture supernatant itself is a waste of cell culture, and its recycling reduces the cost of cell culture.

DRAWINGS

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a cell diagram of a medium composition screening process in a kit of the present invention, wherein Fig. 1A is a cell morphology of a high concentration β-mercaptoethanol medium after 4 hours of cell inoculation, and Fig. 1B is a low concentration bFGF medium inoculation 24 After 1 hour, the cell morphology, Figure 1C is the cell morphology of the cultured cells in the high concentration bFGF medium, and Figure 1D shows the morphology of the culture medium in the low concentration culture supernatant. Figure 1E shows the culture medium of the high concentration culture supernatant. Cell morphology.

Figure 2 is a picture of culturing umbilical cord mesenchymal stem cells using a serum-free medium in the kit of the present invention, wherein Figure 2A shows the cell morphology after 2 hours of inoculation, Figure 2B shows the cell morphology after 24 hours of inoculation, and Figure 2C shows the inoculation. Cell morphology after 48 hours.

Figure 3 is a result of a multiplication curve of hUC-MSC continuous passage using serum-free medium in the kit of the present invention and other media, showing that hUC-MSC cultured in the serum-free medium of the present invention can still be used in long-term culture. Maintain a high rate of proliferation. SCL-M1 and SCL-M2 in the figure are the serum-free medium group of the present invention, FBS-1 and FBS-2 are serum addition groups, and SR-1 and SR-2 are commercially available serum substitute addition groups.

Fig. 4 is a result of analyzing the cell viability and growth characteristics of the obtained umbilical cord mesenchymal stem cells by Vi-Cell cell viability analyzer, wherein Fig. 4A is a real-time activity analysis of hUC-MSCs, and Fig. 4B is a diameter distribution map of hUC-MSC. The results showed that the activity of hUC-MSC was above 99%, and the cell diameter was distributed around 9-15 μm.

Figure 5 (5A to 5I) shows the results of flow cytometry analysis of cell surface molecules, showing that the positive proportion of hUC-MSC expressing CD29, CD44, CD73, CD90, CD105, HLA-ABC is greater than 99%; expression of CD45, CD34 The positive proportion of HLA-DR is less than 1%.

Figure 6 is a result of directed differentiation of hUC-MSCs obtained into osteoblasts and osteoblasts, wherein Fig. 6A shows a dark red compound produced by the color reaction of alizarin red with the calcium nodules of the osteogenesis process. 6B shows that Oil Red O specifically stains for adipocytes in adipocytes.

The best way to implement the invention

The present invention will be further described in detail below with reference to specific embodiments, and the implementation is given. The examples are only illustrative of the invention and are not intended to limit the scope of the invention.

If no specific conditions are specified, it is carried out according to the conventional conditions in the field to which the present invention pertains or the recommended conditions of the instrument reagent supplier; if the source of the commercial source is not indicated, it is a conventional product commercially available.

The mesenchymal stem cell culture supernatant used in the examples was prepared as follows:

Human umbilical cord mesenchymal stem cells isolated from fresh umbilical cord tissue of healthy newborns born or cesarean section were inoculated into T175 cell culture flask at a density of 2×10 ⁴ cells/cm ² , and 25 ml serum-free medium was added. After culturing for 48 hours (the cells reached 70% confluence), fresh umbilical cord mesenchymal stem cell culture supernatant was collected; wherein the serum-free medium contained 0.1 part by volume of β-mercaptoethanol and 1 part by volume of an aqueous solution of non-essential amino acids (11140, Gibco), 94 parts by volume of a-MEM/DMEM-F12 and recombinant human basic fibroblast growth factor at a final concentration of 10 ng/ml;

The cells suspended in the culture supernatant and the cell debris were removed by centrifugation at 3000 g for 30 min at 4 °C;

The supernatant obtained by centrifugation was transferred to a new centrifuge tube, and centrifuged at 10000 g for 50 min at 4 ° C to remove cytoplasm and other impurities in the supernatant;

The supernatant was recovered and sterilized by a 0.22 μm microfiltration membrane;

The filtrate was transferred to a 3 KD ultrafiltration concentrating tube, centrifuged at 3000-4500 g for 80 min at 4 ° C, and the filtrate was concentrated to 1/30 of its volume;

The final volume was adjusted to 1/20 of the initially collected cell culture supernatant with PBS buffer.

Example 1 Screening of medium composition in the kit of the present invention

(1) Screening of β-mercaptoethanol content

Test medium: 0.01, 0.02, 0.05, 0.1, 0.15, 0.2, 0.3 or 0.5 parts by volume of β-mercaptoethanol, 10 ng/ml of recombinant human basic fibroblast growth factor (b-FGF, Peprotech), 1 A volume of a non-essential amino acid aqueous solution (11140, Gibco), 5 parts by volume of a culture supernatant concentrate, and 94 parts by volume of a-MEM.

In the biosafety cabinet, the third generation of hUC-MSC isolated from the natural umbilical cord of the newborn baby was inoculated into the T75 cell culture flask at a density of 2×10 ⁴ cells/cm ² and added 12-15 ml. The medium was tested and the growth of the cells was observed.

RESULTS: Two concentrations of 0.01 and 0.02 parts by volume of β-mercaptoethanol were contained in the medium, respectively. In the group, the cell adherence rate was slow. After 4 hours of inoculation, some cells were still not adherent. After about 8 hours, the cells were all adherent; in the medium, 0.05, 0.1, 0.15 and 0.2 parts by volume, respectively. In the four concentration groups of β-mercaptoethanol, the cells were completely adherent after 4 hours of inoculation, the cells were bright, and the antennae were extended; in the concentration group containing 0.3 and 0.5 volume parts of β-mercaptoethanol, respectively, in the medium. After 4 hours of inoculation, the cells were also adherent, but some of the cells became in a state of deterioration, and early symptoms of differentiation appeared (see Fig. 1A).

(II) Screening of recombinant human basic fibroblast growth factor

Test medium: 0.1 part by volume of β-mercaptoethanol, 1, 2, 5, 8, 10, 12, 15, 18 or 20 ng/ml of recombinant human basic fibroblast growth factor (b-FGF, Peprotech) 1 part by volume of a non-essential amino acid aqueous solution (11140, Gibco), 5 parts by volume of a culture supernatant concentrate, and 94 parts by volume of a-MEM.

Refer to part (a) of the method, inoculate with the same cell source, the same density, add 12-15mL of test medium. Observe the growth of the cells.

RESULTS: In the two concentration groups containing 1, 2 ng/ml bFGF in the medium, the cells proliferated slowly, the cell state was poor, and the state of undernutrition was present (see Figure 1B); in the medium, 5, 8, and 10, respectively. In the concentration group of 12, 15 ng/ml bFGF, the cells grew normally, the brightness was high, and the growth was good. In the concentration group containing 18, 20 ng/ml bFGF in the medium, the cells proliferated well and bright, but after many passages. During the process, the cells are easily differentiated, the cells are aggregated, or the antennae become longer (see Figure 1C).

(3) Screening of the content of the culture supernatant

Test medium: 0.1 part by volume of β-mercaptoethanol, 10 ng/ml of recombinant human basic fibroblast growth factor (b-FGF, Peprotech), 1 part by volume of aqueous solution of non-essential amino acids (11140, Gibco), 1, 2, 4, 5, 6, 8 or 10 parts by volume of the culture supernatant concentrate, 94 parts by volume of a-MEM.

RESULTS: In the two concentration groups containing 1 and 2 volumes of the culture supernatant concentrate in the culture medium, the cells proliferated slowly, and the cells were observed 48 hours after the inoculation, and the cells of hUC-MSC were aggregated to about 60% confluence. The cells are flat and stop proliferating (see Figure 1D); In the three concentration groups of 4, 5, and 6 parts by volume of the culture supernatant, the cells grew well. After 24 hours of inoculation, the cells were observed. The hUC-MSCs were spindle-shaped and vortex-like, with high elongation and bright cells. The degree was 40-60%. After 48 hours of inoculation, the cells were observed. The hUC-MSC cells were bright and reached more than 90% confluence. In the medium, 8 and 10 volumes of the culture supernatant were respectively concentrated in two concentration groups. Local uneven aggregation was observed, and floating objects appeared in the culture flask, and some cells died (see Fig. 1E).

Example 2 Preparation and application of serum-free medium in the kit of the present invention

Serum-free medium preparation:

Formulation: 0.1 part by volume of β-mercaptoethanol, 1 part by volume of a non-essential amino acid aqueous solution (11140, Gibco), 5 parts by volume of the prepared culture supernatant concentrate, 94 parts by volume of a-MEM/DMEM-F12, and Recombinant human basic fibroblast growth factor at a final concentration of 10 ng/ml.

A premix of β-mercaptoethanol, an aqueous solution of non-essential amino acids, and a-MEM/DMEM-F12 was prepared, and the culture supernatant and the recombinant human basic fibroblast growth factor were mixed with the premix.

Cell culture: In the biosafety cabinet, the third generation of hUC-MSCs isolated from the natural umbilical cord of the newborn baby was inoculated into the T175 cell culture flask at a density of 2×10 ⁴ cells/cm ² . 15 mL of the serum-free medium of the present invention was transferred to a 37 ° C incubator with a CO ₂ concentration of 5%. After 2 hours of inoculation, the cells were observed to adhere to the cells, and the culture was continued. After about 4 hours, the cells were completely adherent; after 24 hours of inoculation, the cells were observed, and the hUC-MSCs were fusiformly vortex-like, with high elongation, bright cells, and confluence. Up to 40-60%; after 48 hours of inoculation, the cells were observed, hUCMSC cells were bright, more than 90% confluence, and cells were cryopreserved by trypsin digestion. See Figure 2 for the results.

Example 3 Preparation and application of serum-free medium in the kit of the present invention

Serum-free medium preparation:

Formulation: 0.05 parts by volume of β-mercaptoethanol, 2 parts by volume of a non-essential amino acid aqueous solution, 4 parts by volume of a culture supernatant concentrate, 90 parts by volume of DMEM-F12, and a recombinant human alkaline form having a final concentration of 15 ng/ml. Fiber growth factor.

A serum-free medium was prepared according to the method of Example 2.

Cell culture. Referring to the method of Example 2, the cells were observed to be in good condition after 24 h of inoculation. The confluence reached 40% and continued to culture. After 48 hours, the cells were in a fusiform vortex, reaching 80%, and the culture was not rolled up.

Example 4 Preparation and application of serum-free medium in the kit of the present invention

Serum-free medium preparation:

Formulation: 0.2 parts by volume of β-mercaptoethanol, 0.5 parts by volume of a non-essential amino acid aqueous solution, 6 parts by volume of a culture supernatant concentrate, 95 parts by volume of a-MEM, and a recombinant human alkaline base having a final concentration of 5 ng/ml. Fiber growth factor.

A serum-free medium was prepared according to the method of Example 2.

Cell culture was carried out according to the method of Example 2, and the cells were basically adhered to the wall for 4 hours after inoculation. The cells were in a bright original shape. After 24 hours of inoculation, the cells were in good condition and began to vortex and merge, and the confluence was 30-50%. The culture was continued. After 48 hours, The cells are bright, the antennae stretch naturally, and the cells meet 80%.

Example 5 Effect of Different Media on Cell Multiplication

Test medium:

Serum-free medium (SCL-M) in the kit of the present invention: 94 parts by volume of a-MEM/DMEM-F12, 0.1 part by volume of β-mercaptoethanol, 1 part by volume of aqueous solution of non-essential amino acids (11140, Gibco) 5 parts by volume of the culture supernatant concentrate and a recombinant human basic fibroblast growth factor at a final concentration of 10 ng/ml;

Serum medium (FBS): 89 parts by volume of a-MEM/DMEM-F12, 0.1 part by volume of β-mercaptoethanol, 1 part by volume of aqueous solution of non-essential amino acids (11140, Gibco), 10 parts by volume of FBS and Recombinant human basic fibroblast growth factor at a final concentration of 10 ng/ml;

Serum-free medium (SR) supplemented with conventional serum replacement: 89 parts by volume of a-MEM/DMEM-F12, 0.1 part by volume of β-mercaptoethanol, 1 part by volume of aqueous solution of non-essential amino acids (11140, Gibco), 10 parts by volume of serum replacement (Gibco product, Cat. No. 10828-010) and recombinant human basic fibroblast growth factor at a final concentration of 10 ng/ml.

The first generation cells cultured in Example 2 were seeded at a density of 2×10 ⁴ cells/cm ² in a 6-well plate, and 2 ml of three test mediums were added to each well. After 48 hours of culture, 6 wells were removed after trypsin digestion. The total number of cells, continue to inoculate 6-well plates at the same density, and repeat the same procedure to 21 passages. Make 2 repetitions for each set of medium.

Cumulative multiplication calculation:

(1) Population doubling (PD) = [log ₁₀ (N _H ) - log ₁₀ (N ₀ )] / log ₁₀ (2); wherein N _H is the number of harvested cells, and N ₀ is the number of cells inoculated.

(2) Cumulative population doubling (CPD) is the sum of successive passages of PD.

As a result, as shown in Fig. 3, in the continuous subculture of hUC-MSCs using the serum-free medium of the present invention, the high-algebra (P21) cells were able to maintain good amplification effects (SCL-M1 and SCL-M2 groups); When the serum medium was subjected to continuous passage, cell proliferation was significantly slowed at the 13th passage (FBS-1 and FBS-2 groups); in the medium supplemented with serum replacement (SR-1 and SR-2 groups) ), cell proliferation is relatively stable, but slower than the other groups.

Example 5 Cell viability analyzer for analyzing cell viability and growth characteristics of hUCMSC

The 3rd generation cells cultured in Example 2 were inoculated into T25 flasks, and after the cells reached 95%-100% confluence, 0.125% trypsinization was performed, and the collected cells were seeded at 2×10 ⁵ cells/well density at two 6 Orifice plate. After the cells were all adherent and partially grown for 10 hours, two wells were collected and 500 μL of PBS was added to prepare a cell suspension, which was analyzed by a cell analysis (cell viability analyzer Vi-Cell XR, Beckman). Samples were taken every 12 hours thereafter and growth curves were plotted.

The results are shown in Figure 4. The hUCMSC activity is above 99.7%. The cell diameter distribution is 9-12μm. The hUCMSCs with shuttle-shaped vortex growth have complete roundness after digestion; and they have latent, logarithmic growth and plateau proliferation. characteristic.

Example 6 Flow cytometry analysis of surface markers of hUCMSC

The third passage cells cultured in Example 2 were taken, and after the cells were grown to 90% confluence, 2 mL of 0.125% trypsin was digested, and then centrifuged at 1200 rpm for 6 minutes at 4 ° C, the supernatant was discarded, and the cells were washed twice, and the cells were washed twice. Transfer 1 × 10 ⁵ tubes to the flow tube, add 5 μL CD34-PE, CD45-FITC, CD29-FITC, CD44-PE, CD73-PE, CD105-PE, CD90-FITC, HLA-ABC-FITC, HLA-DR-PE, IgG1-PE (isotype control) and IgG1-FITC (isotype control) antibody, mix at 4 ° C for 30 minutes in the dark, wash once with PBS, centrifuge to remove the supernatant, add 500 μL PBS and resuspend, On-machine detection (flow cytometry XL, Beckman), 1 x 10 ⁴ cells were collected per sample. The results are shown in Figure 5, and the cellular immunophenotype is as follows:

Positive expression (greater than 99%): CD29, CD44, CD73, CD105, CD90, HLA-ABC;

Negative expression (less than 1%): CD34, CD45, HLA-DR.

Example 7 Identification of multidirectional differentiation potential of hUC-MSC

1) Osteogenic differentiation

The 3rd generation hUCMSC cultured in Example 2 was inoculated to a 6-well cell culture plate at 3 × 10 ⁴ cells/cm ² , and 24 hours later, freshly prepared human UC MSC osteogenic differentiation medium (HUXUC-) was added per well. 90021, Saiye products) 2mL, after which every 3 days to replace the fresh osteogenic differentiation induction medium, 2 weeks after the paraformaldehyde fixation, alizarin red staining 3-5min.

The results are shown in Fig. 6A, which shows that hUCMSCs cultured in the medium of the present invention undergo a deep red color reaction with calcium nodules of the osteogenesis process two weeks after osteogenic induction.

2) Adipogenic differentiation

The third generation hUCMSC cultured in Example 2 was inoculated to a 6-well cell culture plate at 2×10 ⁴ cells/cm ² , and after the cells reached 100% confluence, an adipogenic differentiation medium A was added per well (HUXUC- 90031, the Saiye product) began to induce, and after 3 days, it was replaced with the adipogenic differentiation medium B for 24 hours, and thus circulated. When the lipid droplets appeared more but smaller, the adipogenic induction solution B was maintained for 7 days, and 4% paraformaldehyde was fixed and oil red O stained after the induction.

The results are shown in Fig. 6B, which shows that the hUCMSCs cultured in the medium of the present invention stained the adipocytes significantly after two weeks of adipogenic induction.

The above description of the specific embodiments of the present invention is not intended to limit the invention, and various modifications and changes may be made by those skilled in the art without departing from the spirit and scope of the invention.

Claims

A kit for culturing cells, characterized in that the kit comprises a serum-free medium containing a-MEM/DMEM-F12, β-mercaptoethanol, non-essential amino acids, recombinant human A culture supernatant of basic fibroblast growth factor (b-FGF) and human umbilical cord mesenchymal stem cells (hUC-MSCs).
The kit according to claim 1, wherein the serum-free medium comprises 0.05 to 0.2 parts by volume of β-mercaptoethanol, 0.5 to 2 parts by volume of an aqueous solution of a non-essential amino acid, and 4 to 6 parts by volume of the culture. a supernatant concentrate, 90-95 parts by volume of a-MEM/DMEM-F12, and a recombinant human basic fibroblast growth factor at a final concentration of 5-15 ng/ml, wherein the non-essential amino acid aqueous solution contains a concentration of 8 -12 mM glycine, alanine, L-aspartamide, L-aspartic acid, glutamic acid, proline and serine;

Preferably, the serum-free medium comprises 0.1 parts by volume of β-mercaptoethanol, 1 part by volume of an aqueous solution of non-essential amino acids, 5 parts by volume of a culture supernatant concentrate, 94 parts by volume of a-MEM/DMEM-F12, and Recombinant human basic fibroblast growth factor at a final concentration of 10 ng/ml.
The kit according to claim 1 or 2, wherein the culture supernatant concentrate is obtained by a method comprising the following steps:

Human umbilical cord mesenchymal stem cell culture supernatant was collected, centrifuged, filtered through a microfiltration membrane, and concentrated by ultrafiltration;

Preferably, the culture supernatant concentrate is obtained by a method comprising the following steps:

(1) Human umbilical cord mesenchymal stem cells are seeded in serum-free medium at a density of 0.5-4×10 4 cells/cm 2 for 48-72 hours to allow cells to reach 70%-90% confluence, and fresh umbilical cords are collected. Mesenchymal stem cell culture supernatant;

(2) Centrifuge at 3000-5000 g for 15-40 min at 4 ° C to remove cells and cell debris suspended in the culture supernatant;

(3) The supernatant obtained in the step (2) is centrifuged at 10000 g for 30-60 min at 4 ° C to remove cytoplasm and other impurities in the supernatant;

(4) The supernatant obtained in the step (3) is sterilized by a 0.22 μm microfiltration membrane;

(5) The filtrate obtained in the step (4) is transferred into a 3KD ultrafiltration concentrating tube, centrifuged at 3000-4500 g for 60-90 min at 4 ° C, and the filtrate is concentrated to 1/20 to 1/ volume. 50;

(6) The concentrate obtained in the step (5) was adjusted to a final volume of PBS buffer to be 1/20 of the cell culture supernatant collected in the step (1).
The kit according to any one of claims 1 to 3, wherein in the step (1), the human umbilical cord mesenchymal stem cells (hUC-MSCs) are from natural childbirth or cesarean section. Human umbilical cord mesenchymal stem cells isolated from fresh umbilical cord tissue of healthy newborns;

Preferably, the serum-free medium in the step (1) contains a-MEM/DMEM-F12, β-mercaptoethanol, non-essential amino acids, recombinant human basic fibroblast growth factor (b-FGF), and optionally A culture supernatant of human umbilical cord mesenchymal stem cells; preferably comprising 0.05-0.2 parts by volume of β-mercaptoethanol, 0.5-2 parts by volume of a non-essential amino acid aqueous solution, optionally 4-6 parts by volume of a culture supernatant concentrated a solution, 90-95 parts by volume of a-MEM/DMEM-F12 and a recombinant human basic fibroblast growth factor at a final concentration of 5-15 ng/ml, wherein the aqueous solution of the non-essential amino acids comprises a concentration of 8-12 mM each. Glycine, alanine, L-aspartamide, L-aspartic acid, glutamic acid, valine, and serine; more preferably, 0.1 part by volume of β-mercaptoethanol, 1 part by volume of a non-essential amino acid aqueous solution, or A selected 5 parts by volume of the culture supernatant concentrate, 94 parts by volume of a-MEM/DMEM-F12, and a recombinant human basic fibroblast growth factor at a final concentration of 10 ng/ml.
The kit according to any one of claims 1 to 4, wherein the serum-free medium is obtained by a preparation method comprising the following steps:

The human umbilical cord mesenchymal stem cell culture supernatant is collected, centrifuged, filtered through a microfiltration membrane, and concentrated by ultrafiltration to obtain a culture supernatant concentrate;

Preparing a premix with β-mercaptoethanol, non-essential amino acids, and a-MEM/DMEM-F12;

The culture supernatant, the premix, and the recombinant human basic fibroblast growth factor are mixed.
The kit according to any one of claims 1 to 5, wherein the culture supernatant concentrate in the preparation method is obtained by a method comprising the following steps:

(1) Human umbilical cord mesenchymal stem cells are seeded in serum-free medium at a density of 0.5-4×10 4 cells/cm 2 for 48-72 hours to allow cells to reach 70%-90% confluence, and fresh umbilical cords are collected. Mesenchymal stem cell culture supernatant;

(2) centrifugation at 3000-4500 g for 15-40 min at 4 ° C to remove cells and cell debris suspended in the culture supernatant;

(3) The supernatant obtained in the step (2) is centrifuged at 10000 g for 40-80 min at 4 ° C. Remove cytoplasm and other impurities from the supernatant;

(4) The supernatant obtained in the step (3) is sterilized by a 0.22 μm microfiltration membrane;

(5) The filtrate obtained in the step (4) is transferred into a 3KD ultrafiltration concentrating tube, centrifuged at 3000-4500 g for 60-90 min at 4 ° C, and the filtrate is concentrated to 1/20 to 1 of its volume. /50;

(6) The concentrate obtained in the step (5) was adjusted to a final volume of PBS buffer to be 1/20 of the cell culture supernatant collected in the step (1).
The kit according to any one of claims 1 to 6, wherein the stem cells are isolated from a mammal, such as a human tissue or organ, which is in bone marrow, umbilical cord and fat. One or more;

Preferably, the stem cells are umbilical cord mesenchymal stem cells of mammalian origin, more preferably human-derived umbilical cord mesenchymal stem cells;

More preferably, the stem cells are human umbilical cord mesenchymal stem cells isolated from fresh umbilical cord tissue of a healthy newborn born or cesarean section.
Use of the kit according to any one of claims 1 to 7 in cell culture;

Preferably, the cell culture is a stem cell culture, preferably a stem cell serum free culture.
The use according to claim 8, wherein the stem cells are isolated from a mammal, such as a human tissue or organ, the tissue or organ being one or more of bone marrow, umbilical cord and fat;

Preferably, the stem cells are umbilical cord mesenchymal stem cells of mammalian origin, more preferably human-derived umbilical cord mesenchymal stem cells;

More preferably, the stem cells are human umbilical cord mesenchymal stem cells isolated from fresh umbilical cord tissue of a healthy newborn born or cesarean section.