WO2017096619A1 - Kit de réactifs pour culture cellulaire - Google Patents

Kit de réactifs pour culture cellulaire Download PDF

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WO2017096619A1
WO2017096619A1 PCT/CN2015/097156 CN2015097156W WO2017096619A1 WO 2017096619 A1 WO2017096619 A1 WO 2017096619A1 CN 2015097156 W CN2015097156 W CN 2015097156W WO 2017096619 A1 WO2017096619 A1 WO 2017096619A1
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umbilical cord
cells
culture supernatant
parts
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PCT/CN2015/097156
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郭镭
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郭镭
里程
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  • 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.
  • 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.
  • hUC-MSCs human Umbilical Cord mesenchymal stem cells 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.
  • hUC-MSCs 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. .
  • 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.
  • kits for culturing cells comprising a serum-free medium containing a concentrate obtained from a cell culture, particularly suitable in a serum-free environment Cultured cells.
  • the invention provides a kit for culturing cells, the kit comprising a serum-free medium comprising a-MEM/DMEM-F12, ⁇ -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).
  • a serum-free medium comprising a-MEM/DMEM-F12, ⁇ -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).
  • the serum-free medium comprises 0.05-0.2 parts by volume of ⁇ -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;
  • the serum-free medium comprises 0.1 parts by volume of ⁇ -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.
  • the culture supernatant concentrate is obtained by a method comprising the following steps:
  • 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.
  • 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;
  • the human umbilical cord mesenchymal stem cells are preferably human umbilical cord mesenchymal stem cells isolated from fresh umbilical cord tissue of a healthy newborn born or cesarean section.
  • the serum-free medium in step (1) contains a-MEM/DMEM-F12, ⁇ -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 ⁇ -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
  • 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;
  • the culture supernatant, the premix, and the recombinant human basic fibroblast growth factor are mixed.
  • the culture supernatant concentrate is preferably obtained by a method comprising the following steps:
  • 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.
  • 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;
  • 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.
  • 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 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.
  • 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.
  • 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 ⁇ -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
  • 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 ⁇ m.
  • Figure 5 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 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.
  • the serum-free medium contained 0.1 part by volume of ⁇ -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 ⁇ m 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.
  • Test medium 0.01, 0.02, 0.05, 0.1, 0.15, 0.2, 0.3 or 0.5 parts by volume of ⁇ -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.
  • b-FGF human basic fibroblast growth factor
  • 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 ⁇ -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 ⁇ -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 ⁇ -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).
  • Test medium 0.1 part by volume of ⁇ -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.
  • b-FGF human basic fibroblast growth factor
  • Test medium 0.1 part by volume of ⁇ -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.
  • b-FGF human basic fibroblast growth factor
  • 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).
  • Serum-free medium preparation
  • Formulation 0.1 part by volume of ⁇ -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 ⁇ -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.
  • Serum-free medium preparation
  • Formulation 0.05 parts by volume of ⁇ -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.
  • 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.
  • Serum-free medium preparation
  • Formulation 0.2 parts by volume of ⁇ -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%.
  • 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 ⁇ -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 89 parts by volume of a-MEM/DMEM-F12, 0.1 part by volume of ⁇ -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 supplemented with conventional serum replacement: 89 parts by volume of a-MEM/DMEM-F12, 0.1 part by volume of ⁇ -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.
  • SR Serum-free medium supplemented with conventional serum replacement: 89 parts by volume of a-MEM/DMEM-F12, 0.1 part by volume of ⁇ -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.
  • Example 2 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.
  • CPD Cumulative population doubling
  • 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 ⁇ L 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 9-12 ⁇ m.
  • the hUCMSCs with shuttle-shaped vortex growth have complete roundness after digestion; and they have latent, logarithmic growth and plateau proliferation. characteristic.
  • 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.
  • Negative expression (less than 1%): CD34, CD45, HLA-DR.
  • 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.
  • Fig. 6A 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.
  • 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.
  • the adipogenic induction solution B was maintained for 7 days, and 4% paraformaldehyde was fixed and oil red O stained after the induction.
  • Fig. 6B shows that the hUCMSCs cultured in the medium of the present invention stained the adipocytes significantly after two weeks of adipogenic induction.

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Abstract

L'invention concerne un nouveau kit de réactifs pour la culture sans sérum de cellules souches, le kit de réactifs comprenant un milieu de culture sans sérum, les ingrédients dudit milieu de culture comprenant : de 0,05 à 2 parties en volume de bêta-mercaptoéthanol, 0,5 à 2 parties en volume d'une solution aqueuse d'acides aminés non-essentiels, de 4 à 6 parties en volume d'un concentré de surnageant de culture de cellules souches mésenchymateuses humaines, et de 90 à 95 parties en volume de milieu α-MEM/DMEM-F12 et du facteur de croissance des fibroblastes FGF2 humain recombiné à une concentration finale de 5,5 ng/mL.
PCT/CN2015/097156 2015-12-11 2015-12-11 Kit de réactifs pour culture cellulaire WO2017096619A1 (fr)

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CN115372233A (zh) * 2022-10-25 2022-11-22 华夏源(上海)生命科技有限公司 一种脂肪干细胞细胞周期的检测方法

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CN115372233A (zh) * 2022-10-25 2022-11-22 华夏源(上海)生命科技有限公司 一种脂肪干细胞细胞周期的检测方法

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