WO2022188395A1 - 一种多能干细胞的培养方法 - Google Patents

一种多能干细胞的培养方法 Download PDF

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WO2022188395A1
WO2022188395A1 PCT/CN2021/120868 CN2021120868W WO2022188395A1 WO 2022188395 A1 WO2022188395 A1 WO 2022188395A1 CN 2021120868 W CN2021120868 W CN 2021120868W WO 2022188395 A1 WO2022188395 A1 WO 2022188395A1
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stem cells
culture
pluripotent stem
cells
culturing
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PCT/CN2021/120868
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French (fr)
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宋益哲
梁德灿
唐婷婷
陈晓倩
欧镇生
李景秋
邢佩雯
刘靖
张世都
郭蕾蕾
郑清炼
叶群瑞
陈小锋
李文佳
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广东东阳光药业有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening

Definitions

  • the invention relates to the field of biotechnology, in particular to a method for culturing pluripotent stem cells.
  • Stem cells are a special cell group with self-renewal and differentiation potential. Under appropriate culture conditions in vitro, stem cells can be massively expanded and differentiated into cells with specific functions. Therefore, stem cells can provide cells needed for transplantation for clinical disease treatment. At the same time, because drug screening and safety testing are not directly carried out in the human body, human stem cells have also become an ideal model for large-scale new drug screening and drug research.
  • stem cells derived from different developmental stages and different tissues and organs have great differences in gene expression regulation, epigenetic status, in vitro proliferation and differentiation potential. Generally speaking, stem cells can be divided into totipotent stem cells, pluripotent stem cells, multipotent stem cells and unipotent stem cells according to their different proliferation and differentiation potentials. stem cells).
  • Pluripotent stem cells lose the ability to develop into a complete individual, but they can differentiate into all cell types of an individual, thereby forming all tissues and organs of the body. Therefore, pluripotent stem cells are widely used in various fields such as tissue differentiation research, drug testing, and regenerative medicine. Especially after the establishment of induced pluripotent stem cells (iPSCs), the research in this field has made remarkable progress.
  • iPSCs induced pluripotent stem cells
  • pluripotent stem cells In the application process of pluripotent stem cells, it is first necessary to maintain the undifferentiated state of pluripotent stem cells for culturing, and then induce differentiation of pluripotent stem cells to obtain target cells.
  • the flat culture differentiation system has the disadvantage that the large-scale expansion of cells is limited, and it is difficult to obtain a sufficient number of cells for research , and the differentiation efficiency is low, and it is impossible to make an accurate assessment of cell growth and differentiation.
  • the large-scale suspension culture system has the disadvantage that the system is large, the cost is high, and large-scale drug screening cannot be performed.
  • the object of the present invention is to provide a method for culturing pluripotent stem cells, which can maintain the good stemness of pluripotent stem cells during the process of proliferation and culture, and has high differentiation efficiency and little difference between experiments during the differentiation process.
  • the differentiation inducers of pluripotent stem cells can be efficiently screened, and the differentiation status can be accurately evaluated.
  • the method for culturing pluripotent stem cells provided by the present invention overcomes the shortcomings in the prior art: the differentiation efficiency of the planar culture differentiation system in the existing screening system is low, the cell growth and differentiation cannot be accurately evaluated, and the large system suspension culture The cost of the system is high, and large-scale drug screening cannot be performed.
  • the present invention provides a method for culturing pluripotent stem cells, using a 48-well plate as a culture vessel, a cell seeding density of 4.5-6.5 ⁇ 10 5 cells/mL, and a culture volume of 0.4-0.6 mL.
  • the rotation speed of the shaker was 170-190 rpm for cultivation.
  • the 48-well plate described in the present invention is also called a 48-well culture plate, a 48-well cell culture plate, etc., and the volume of each well is about 1.7 mL, and a commercially available common 48-well plate can be selected, such as Thermo Scientific 48-well plate sold by Nunclon (Cat. No. 150687), 48-well plate sold by Corning (Cat. No. 3548), etc.
  • the culture of pluripotent stem cells often adopts flat culture or large-scale system suspension culture, and there are few reports on the use of multi-well plates; moreover, due to the characteristics of pluripotent stem cells, the culture of pluripotent stem cells cannot be directly applied to other differentiated cells.
  • Cell culture systems or methods for example, in the prior art, some differentiated cells can be cultured in 24-well plates, 48-well plates or 96-well plates according to some general guidelines, but these culture methods are not suitable for many cells.
  • Culture of competent stem cells In the present invention, 96-well plates and 48-well plates are respectively used for culture in the research process, and the relevant conditions such as seeding density, culture volume, shaking speed and the like are simultaneously explored.
  • the cell seeding density is about 4.5 ⁇ 10 5 cells/mL, 5.5 ⁇ 10 5 cells/mL, 6.5 ⁇ 10 5 cells/mL, and the like.
  • the cells are seeded at a density of 5.5 ⁇ 10 5 cells/mL.
  • the culture system is 400 ⁇ L, 500 ⁇ L, 600 ⁇ L, and the like.
  • the rotational speed of the shaker is 170 rpm, 180 rpm, 190 rpm, or the like.
  • the coordination of culture vessel, cell seeding density, culture volume and shaker rotation speed will have a comprehensive impact on cell culture.
  • the culture volume is 0.4-0.6 mL and the rotating speed of the shaker is 170-190 rpm, it can be ensured that the culture medium between each well will not be cross-contaminated; if the corresponding changes are made
  • the culture volume and the rotation speed of the shaker such as reducing the culture volume and increasing the rotation speed of the shaker, or increasing the culture volume and reducing the rotation speed of the shaker, can still keep no cross-contamination between wells. In both cases, the aggregate morphology of cells and the uniformity of cell particle size will be negatively affected, and in severe cases, even the cells after culture cannot maintain stemness.
  • pluripotent stem cells basically conforms to the general knowledge of those skilled in the art. For example, increasing the cell seeding density and increasing the rotation speed of the shaker are conducive to the rapid growth of cells , increasing the culture volume and appropriately increasing the rotation speed are beneficial to cell suspension.
  • morphology such as aggregate morphology, uniformity of cell size, etc.
  • quality such as stemness maintenance, etc.
  • the seeding density is 4.5-6.5 ⁇ 10 5 cells/mL
  • the culture volume is 400-600 ⁇ L
  • the shaking speed is 170-190 rpm , can significantly negatively affect the morphology and/or quality of pluripotent stem cells.
  • the method for culturing pluripotent stem cells of the present invention adopts the following culture conditions: the culture temperature is 36.6 ⁇ 0.5°C, the relative humidity is 90 ⁇ 5%, and the CO 2 concentration is 5% (v/v).
  • fresh medium is replaced every 20-28 hours; preferably, fresh medium is replaced every 24 hours.
  • the method for culturing pluripotent stem cells according to the present invention comprises the following steps:
  • step (S2) taking the cell mass suspension obtained by culturing in step (S1) and preparing it as a single cell suspension;
  • step (S3) taking the single cell suspension prepared in step (S2), and repeating steps (S1) and (S2) at least once for culturing.
  • step (S1) culture is performed for 1-5 days.
  • the specific time can be determined according to the type of cells to be cultured, such as 1d, 2d, 3d, 4d or 5d of culture.
  • step (S2) includes: taking the cell mass suspension obtained by culturing in step (S1), and sequentially performing enrichment cell mass, digestion, filtration, centrifugation, resuspension, and optional cell counting, to prepare The single cell suspension.
  • the number of repetitions is an integer selected from 1-15.
  • the number of repetitions is the number of generations of subculture, which can be determined according to the type of cells to be cultured and their growth and differentiation conditions.
  • the number of repetitions of steps (S1) and (S2) is 1 or 2 , 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times or 15 times, etc.
  • the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells (iPS).
  • the embryonic stem cells are human embryonic stem cells.
  • the second aspect of the present invention provides the application of the method for culturing pluripotent stem cells in any aspect of A1), A2), and A3):
  • A2) induce differentiation of the pluripotent stem cells
  • the present invention overcomes the prejudice of the prior art, adopts 48-well plate for culturing pluripotent stem cells for the first time, and establishes a 48-well plate suspension culture system suitable for the growth and differentiation of pluripotent stem cells from scratch, and the culture system Differentiation inducers of pluripotent stem cells can be efficiently screened.
  • the culture system and method for pluripotent stem cells provided by the present invention overcome the low differentiation efficiency of the flat culture and differentiation system in the prior art, the inability to accurately assess the growth and differentiation of cells, and the high cost of the large-scale suspension culture system and the inability to Insufficient to carry out large-scale drug screening.
  • the technical solution of the present invention has the following significant advantages: (1) the pluripotent stem cells cultured according to the present invention can maintain a good cell shape and a stable multiplication multiple, so as to obtain a larger number of cells for research; (2) culture After several generations, pluripotent stem cells can still maintain a normal karyotype and a better cell phenotype; (3) the size and shape of the cell mass in the cultured pluripotent stem cells are basically the same during the differentiation process, reducing the differences between experiments; (4) ) can use this system for high-throughput drug screening, avoiding the use of expensive high-throughput analytical instruments, resulting in significant cost savings.
  • Figure 1 Human embryonic stem cells H9 were cultured according to a certain culture method of the present invention (seeding density 5.5 ⁇ 10 5 cells/mL, culture system 600 ⁇ L, rotating speed 180 rpm), and the results of cell aggregation morphology detection;
  • Figure 2 Human embryonic stem cells H9 were cultured according to a certain culture method of the present invention (inoculation density 5.5 ⁇ 10 5 cells/mL, culture system 600 ⁇ L, rotating speed 180 rpm), and immunofluorescence detection results;
  • Figure 3 Human embryonic stem cells H9 were cultured according to a certain culture method of the present invention (seeding density 5.5 ⁇ 10 5 cells/mL, culture system 600 ⁇ L, rotating speed 180 rpm), and flow cytometry detection results;
  • Figure 4 Human embryonic stem cells H9 were cultured according to a certain culture method of the present invention (seeding density 5.5 ⁇ 10 5 cells/mL, culture system 600 ⁇ L, rotating speed 180 rpm), and the karyotype detection results of the cells;
  • FIG. 5 Induced pluripotent stem cells (iPS) were cultured according to a certain culture method of the present invention (seeding density 5.5 ⁇ 10 5 cells/mL, culture system 600 ⁇ L, rotating speed 180 rpm), and the results of cell aggregation morphology detection;
  • FIG. 6 Induced pluripotent stem cells (iPS) were cultured according to a certain culture method of the present invention (seeding density 5.5 ⁇ 10 5 cells/mL, culture system 600 ⁇ L, rotation speed 180 rpm), and flow cytometry detection results;
  • FIG. 7 Induced pluripotent stem cells (iPS) were cultured according to a certain culture method of the present invention (seeding density 5.5 ⁇ 10 5 cells/mL, culture system 600 ⁇ L, rotating speed 180 rpm), and the results of cell karyotype detection;
  • Figure 8 Human embryonic stem cells H9 were cultured according to a certain culture method of the present invention (seeding density 4.5 ⁇ 10 5 cells/mL, culture system 400 ⁇ L, rotating speed 180 rpm), and the results of cell aggregation morphology detection;
  • Figure 9 Human embryonic stem cells H9 were cultured according to a certain culture method of the present invention (seeding density 4.5 ⁇ 10 5 cells/mL, culture system 500 ⁇ L, rotating speed 180 rpm), and the results of cell aggregation morphology detection;
  • Figure 10 Human embryonic stem cells H9 were cultured according to a certain culture method of the present invention (seeding density 4.5 ⁇ 10 5 cells/mL, culture system 600 ⁇ L, rotating speed 180 rpm), and the results of cell aggregation morphology detection;
  • Figure 11 Human embryonic stem cells H9 were cultured according to a certain culturing method of the present invention (seeding density 6.5 ⁇ 10 5 cells/mL, culture system 600 ⁇ L, rotating speed 180 rpm), and the results of cell aggregation morphology detection;
  • Figure 12 Human embryonic stem cells H9 were cultured according to a certain culture method of the present invention (seeding density 5.5 ⁇ 10 5 cells/mL, culture system 600 ⁇ L, rotating speed 170 rpm), and the results of cell aggregation morphology detection;
  • Figure 13 Human embryonic stem cells H9 were cultured according to a certain culture method of the present invention (seeding density 5.5 ⁇ 10 5 cells/mL, culture system 600 ⁇ L, rotating speed 190 rpm), and the results of cell aggregation morphology detection;
  • FIG. 14 Culture and induction of differentiation of human embryonic stem cells H9 using the culture method of the present invention, the analysis results of the later screening of SANT1 analogous chemical library;
  • Figure 15 Culturing and inducing differentiation induced pluripotent stem cells (iPS) using the culture method of the present invention, the analysis results of the later screening of LDN1931189 analogous chemical library;
  • Figure 16 Human embryonic stem cells H9 were cultured in 96-well plates with different seeding densities, and the results of cell aggregation morphology detection;
  • Figure 17 Human embryonic stem cells H9 were cultured in 96-well plates with different seeding densities, and the results of immunofluorescence detection;
  • Figure 18 Human embryonic stem cells H9 were cultured in 96-well plates with different seeding densities, and the results were detected by flow cytometry;
  • Figure 19 Human embryonic stem cells H9 were cultured in 96-well plates with different seeding densities, and the results of cell karyotype detection;
  • Figure 20 Human embryonic stem cells H9 were cultured according to a certain culture method of the comparative example (seeding density 4.5 ⁇ 10 5 cells/mL, culture system 700 ⁇ L, rotating speed 180 rpm), and the results of cell aggregation morphology detection;
  • Figure 21 Human embryonic stem cells H9 were cultured according to a certain culture method of the comparative example (seeding density 3.5 ⁇ 10 5 cells/mL, culture system 600 ⁇ L, rotating speed 180 rpm), and the results of cell aggregation morphology detection;
  • Figure 22 Human embryonic stem cells H9 were cultured according to a certain culture method of the comparative example (seeding density 5.5 ⁇ 10 5 cells/mL, culture system 600 ⁇ L, rotation speed 160 rpm), and the results of cell aggregation morphology detection.
  • mTeSR1 complete medium mTeSR 1 Basal Medium, 5 ⁇ mTeSR 1 Supplement
  • DE medium MCDB131 Basal Medium, 1000xAct A, 1000x CHIR99021
  • PGT medium MCDB131 Basal Medium, 2000 ⁇ KGF
  • PP1 differentiation medium MCDB131 Basal Medium, 2000 ⁇ KGF, 10000 ⁇ RA, 10000 ⁇ SANT1, 10000 ⁇ LDN
  • KSR medium 15% KSR, KO DMEM, L-glutamine, 100 ⁇ non-essential amino acids (NEAA), 1000 ⁇ -mercaptoethano
  • NIM medium DMEM/F12, 100 ⁇ N2sup-Plement, 50 ⁇ B27supplement, 100 ⁇ Glutamax, 100 ⁇ NEAA(Gibco), 0.2Mm ascorbic acid
  • the dynamic suspension culture of human embryonic stem cells H9 is carried out, and the related detection of the cultured cells is carried out.
  • the medium is mTeSR1 complete medium, the culture system is 600 ⁇ L, and the rotation speed is 180 rpm. , the temperature is 37°C, the relative humidity is 90 ⁇ 5%, and 5% CO 2 (v/v); the medium is replaced with fresh medium every 24 hours of culture, and the co-culture is carried out for 4 days.
  • the particle size of the cell mass is 300-400 ⁇ m. shown.
  • step (3) Take the cell suspension counted in step (2), repeat the steps described in steps (1) and (2) for subculture, and co-culture for 4 generations.
  • step (1) Collect the cell mass obtained in step (1) in a 1.5 mL EP tube, rinse with PBS three times, add 1 mL of 4% formaldehyde fixative solution for 1 h at room temperature, dehydrate through low- to high-concentration ethanol, and incubate 1 mL of xylene at room temperature for 5 min , repeated 3 times for transparency and air-drying; transfer the cell mass into paraffin and immerse it in paraffin, trim, slice and remove the embedded samples, and bake the obtained slices in a 42°C oven overnight.
  • step (1) Collect the cell mass obtained in step (1) and prepare it as a single cell suspension. Take 3 ⁇ 10 6 cells into an EP tube, wash 2 times with PBS, add 1 mL Fixation Buffer (BD) to fix for 20 min, 1 ⁇ Perm/Wash After washing twice with Buffer (BD), it was divided into 3 equal parts, which were used as unstain group, isotype group and stain group respectively. Among them, 20 ⁇ L, anti-Oct3/4, anti-SSEA-1, anti-SSEA-4 were added to the stain group; 20 ⁇ L PerCP-Cy5.5Mouse IgG1, PE Mouse IgM, Alexa were added to the isotype group 647 Mouse IgG3; unstain not processed.
  • BD Fixation Buffer
  • Collect the cell mass after 4 generations of culture prepare it as a single cell suspension, inoculate it into a T25 cell culture flask at 2 ⁇ 10 5 cells/cm 2 , and transfer it to a third-party institution for karyotyping when the cells reach the logarithmic growth phase.
  • the karyotype test report is shown in Figure 4. According to the test report shown in Figure 4, the karyotype of the cells was normal after 4 passages of culture.
  • iPS induced pluripotent stem cells
  • iPS Induced pluripotent stem cells
  • Figure 5 shows the aggregated morphology of the cells cultured in step (1);
  • Figure 6 shows the results of flow detection. Indicates that good dryness is maintained.
  • Figure 7 the karyotype test report of the cells is shown in Figure 7. According to the test report shown in Figure 7, the karyotype of the cells after 4 generations of culture is normal.
  • the dynamic suspension culture of human embryonic stem cells H9 is carried out, and the relevant detection of the cultured cells is carried out:
  • Human embryonic stem cells H9 were inoculated in a 48-well plate at a seeding density of 4.5 ⁇ 10 5 cells/mL, and the dynamic suspension culture was performed on an orbital shaker.
  • the medium was mTeSR1 complete medium, the culture system was 400 ⁇ L, the speed was 180 rpm, and the temperature was 37 °C, relative humidity 90 ⁇ 5%, 5% CO 2 (v/v); fresh medium was replaced every 24 hours of culture, and co-culture was carried out for 4 days.
  • the aggregated morphology of the cultured cells is shown in Figure 8, and it can be seen that the size of the cell aggregates is uniform and the shape is relatively round.
  • the dynamic suspension culture of human embryonic stem cells H9 is carried out, and the relevant detection of the cultured cells is carried out:
  • Human embryonic stem cells H9 were inoculated in a 48-well plate at a seeding density of 4.5 ⁇ 10 5 cells/mL, and the dynamic suspension culture was carried out on an orbital shaker. °C, relative humidity 90 ⁇ 5%, 5% CO 2 (v/v); fresh medium was replaced every 24 hours of culture, and co-culture was carried out for 4 days.
  • the aggregated morphology of the cultured cells is shown in Figure 9, and it can be seen that the size of the cell aggregates is uniform and the shape is relatively round.
  • Human embryonic stem cells H9 were inoculated in a 48-well plate at a seeding density of 4.5 ⁇ 10 5 cells/mL, and the dynamic suspension culture was carried out on an orbital shaker.
  • the medium was mTeSR1 complete medium, the culture system was 600 ⁇ L, the speed was 180 rpm, and the temperature was 37 °C, relative humidity 90 ⁇ 5%, 5% CO 2 (v/v); fresh medium was replaced every 24 hours of culture, and co-culture was carried out for 4 days.
  • the aggregated morphology of the cultured cells is shown in Figure 10, and it can be seen that the cell aggregates are uniform in size and round in shape.
  • Human embryonic stem cells H9 were seeded in a 48-well plate at a seeding density of 6.5 ⁇ 10 5 cells/mL, and were cultured in dynamic suspension on an orbital shaker.
  • the medium was mTeSR1 complete medium, the culture system was 600 ⁇ L, the speed was 180 rpm, and the temperature was 37 °C, relative humidity 90 ⁇ 5%, 5% CO 2 (v/v); fresh medium was replaced every 24 hours of culture, and co-culture was carried out for 4 days.
  • the aggregated morphology of the cultured cells is shown in Figure 11, and it can be seen that the size of the cell aggregates is relatively uniform and the shape is round.
  • Human embryonic stem cells H9 were inoculated in a 48-well plate at a seeding density of 5.5 ⁇ 10 5 cells/mL, and the dynamic suspension culture was performed on an orbital shaker.
  • the medium was mTeSR1 complete medium, the culture system was 600 ⁇ L, the speed was 170 rpm, and the temperature was 37 °C, relative humidity 90 ⁇ 5%, 5% CO 2 (v/v); fresh medium was replaced every 24 hours of culture, and co-culture was carried out for 4 days.
  • the aggregated morphology of the cultured cells is shown in Figure 12. It can be seen that the size of the cell aggregates is relatively uniform and the shape is round.
  • Human embryonic stem cells H9 were inoculated in a 48-well plate at a seeding density of 5.5 ⁇ 10 5 cells/mL, and the dynamic suspension culture was carried out on an orbital shaker. °C, relative humidity 90 ⁇ 5%, 5% CO 2 (v/v); fresh medium was replaced every 24 hours of culture, and co-culture was carried out for 4 days.
  • the aggregated morphology of the cultured cells is shown in Figure 13, and it can be seen that there are individual larger cell aggregates, but most of the cell aggregates still have good homogeneity and the shape is relatively round.
  • human embryonic stem cells H9 were induced to differentiate into pancreatic progenitor cells, and induction agents were screened.
  • Human embryonic stem cells H9 were inoculated in a 48-well plate at a seeding density of 5.5 ⁇ 10 5 cells/mL, and the dynamic suspension culture was carried out on an orbital shaker.
  • the medium was mTeSR1 complete medium, the culture system was 600 ⁇ L, the speed was 180 rpm, and the temperature was 37 °C, relative humidity 90 ⁇ 5%, 5% CO 2 (v/v); fresh medium was replaced every 24 hours of culture, and co-culture was carried out for 3 days. Then replace the medium to continue culturing, specifically: replace the medium with DE medium (S1) for 3 days, then replace the medium with PGT medium (S2) for 2 days, and then replace the medium with PP1 differentiation medium ( S3) continue to cultivate.
  • DE medium DE medium
  • PGT medium S2
  • S3 replace the medium with PP1 differentiation medium
  • Pre-screening During the above-mentioned culture process, when the PP1 differentiation medium (S3) was replaced, the library was divided into groups, and 10 ⁇ M of different SANT1-like chemicals were added to each group, and the group added with 0.25 ⁇ M SANT1 was used as a positive control group. After culturing with PP1 differentiation medium (S3) for 2 days, the cells of each group were detected by Beckman flow cytometer to analyze the expression of PDX1. The early hit screenings are shown in Table 1.
  • the hit candidate chemicals screened in the early stage of Table 1 were added to the PP1 differentiation medium (S3) at the working concentration of 0 ⁇ M, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M and 50 ⁇ M respectively, and the obtained medium was used to culture the original intestinal tube cells, On day 3 of differentiation, cells in 48-well plates were transferred to 96-well plates to fix and analyze the expression of PDX1.
  • the analysis results are shown in FIG. 14 . According to the analysis results in FIG. 14 , 5 ⁇ M cyclopamine has a good substitution effect and can be used as an inducer for inducing the differentiation of human embryonic stem cells H9 into pancreatic progenitor cells.
  • iPS induced pluripotent stem cells
  • Induced pluripotent stem cells were seeded in a 48-well plate at a seeding density of 5.5 ⁇ 10 5 cells/mL, and the dynamic suspension culture was carried out on an orbital shaker.
  • the medium was mTeSR1 complete medium, the culture system was 600 ⁇ L, and the rotation speed was 180rpm, temperature 37°C, relative humidity 90 ⁇ 5%, 5% CO 2 (v/v); fresh medium was replaced every 24 hours of culture, and co-culture was carried out for 3 days. Then replace the medium to continue the culture, specifically: replace the medium with the differentiation medium (S1) for 6 days, then replace the medium with the KSR medium (S2) for 3 days, and then replace the medium with the NIM medium (S3) ) cultured for 5d.
  • S1 differentiation medium
  • S2 KSR medium
  • S3 NIM medium
  • the library was set up when it was replaced with NIM medium (S3), and 10 ⁇ M of different LDN1931189-like chemicals were added to each group, and the group added with 5.0 ⁇ M LDN1931189 was used as a positive control group. After culturing with NIM medium (S3) for 5 days, the cells in each group were detected by Beckman flow cytometer to analyze the expression of OTX1/2. The early hit screenings are shown in Table 2.
  • Late stage The hit candidate chemicals screened in the early stage of Table 2 were added to the NIM medium (S3) at the working concentration of 0 ⁇ M, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M, and 50 ⁇ M, respectively, and the obtained medium was used to culture the cortical progenitor cells , when differentiated to the 6th day, the cells in the 48-well plate were transferred to the 96-well plate for fixation and the expression of OTX1/2 was analyzed.
  • the analysis results are shown in Figure 15. According to the analysis results in FIG. 15 , 10 ⁇ M of DMH-1 has a good substitution effect and can be used as an inducer for inducing the differentiation of induced pluripotent stem cells (iPS) into neural cells.
  • iPS induced pluripotent stem cells
  • the aggregated morphology of the cells cultured in step (1) is shown in Figure 16, and it was found that there were obvious differences in particle size; after comprehensive comparison, the group with a cell number of 3000 cells/well was selected for subsequent immunofluorescence detection and flow detection. and karyotyping.
  • the results of immunofluorescence detection are shown in Figure 17. It can be seen that the cells cultured in this system do not express significantly SSEA4, and the stemness of pluripotent stem cells cannot be well maintained.
  • the results of flow cytometry are shown in Figure 18. According to Figure 18, the Oct4/SSEA4 positive cell group appeared tailing and grouping, indicating that the cultured cells were not pure and had other cells. After 4 generations of culture, the karyotype detection report of the cells is shown in Figure 19. According to the detection report shown in Figure 19, the karyotype of the cells after 4 generations of culture is abnormal.
  • Human embryonic stem cells H9 were inoculated in a 48-well plate at a seeding density of 4.5 ⁇ 10 5 cells/mL, and the dynamic suspension culture was performed on an orbital shaker.
  • the medium was mTeSR1 complete medium, the culture system was 700 ⁇ L, the speed was 180 rpm, and the temperature was 37 °C, relative humidity 90 ⁇ 5%, 5% CO 2 (v/v); fresh medium was replaced every 24 hours of culture, and co-culture was carried out for 4 days.
  • the aggregated morphology of the cultured cells is shown in Figure 20. It can be seen that when the culture volume is increased to 700 ⁇ L, the cell aggregates are of different sizes, and a significantly larger cell mass appears.
  • Human embryonic stem cells H9 were inoculated in a 48-well plate at a seeding density of 3.5 ⁇ 10 5 cells/mL, and the dynamic suspension culture was carried out on an orbital shaker.
  • the medium was mTeSR1 complete medium, the culture system was 600 ⁇ L, the speed was 180 rpm, and the temperature was 37 °C, relative humidity 90 ⁇ 5%, 5% CO 2 (v/v); fresh medium was replaced every 24 hours of culture, and co-culture was carried out for 4 days.
  • the aggregated morphology of the cultured cells is shown in Figure 21. It can be seen that the size uniformity of the cell aggregates is very poor, and there is a phenomenon of fusion.
  • Human embryonic stem cells H9 were inoculated in a 48-well plate at a seeding density of 5.5 ⁇ 10 5 cells/mL, and the dynamic suspension culture was carried out on an orbital shaker.
  • the medium was mTeSR1 complete medium, the culture system was 600 ⁇ L, the speed was 160 rpm, and the temperature was 37 °C, relative humidity 90 ⁇ 5%, 5% CO 2 (v/v); fresh medium was replaced every 24 hours of culture, and co-culture was carried out for 4 days.
  • the aggregated morphology of the cultured cells is shown in Fig. 22. It can be seen that the number of cells is small, and the cells cannot form agglomerates well, and the particle size of the cell clusters is not uniform.

Abstract

一种多能干细胞的培养方法，采用48孔板为培养容器，细胞接种密度为4.5～6.5×10 5细胞/mL，培养体积为0.4～0.6mL，采用摇床转速为170～190rpm进行培养。

Description

一种多能干细胞的培养方法 技术领域
本发明涉及生物技术领域，具体涉及一种多能干细胞的培养方法。
背景技术
干细胞是一类具有自我更新和分化潜能的特殊细胞类群。在体外适当的培养条件下，干细胞可以被大量扩增和分化为具有特定功能的细胞。因此，干细胞可以为临床疾病治疗提供移植所需的细胞。同时，由于药物筛选和安全性检验不直接在人体进行，人的干细胞也成为大规模的新药筛选和药物研究的理想模型。
来源于不同发育阶段和不同组织器官的干细胞在基因表达调控、表观遗传状态、体外增殖和分化潜能等方面都存在很大的差异。一般来讲，根据干细胞增殖能力和分化潜能的不同，可以把干细胞分为全能干细胞(totipotent stem cell)、多能干细胞(pluripotent stem cell)、专能干细胞(multipotent stem cell)和单能干细胞(unipotent stem cell)。
多能干细胞失去了发育为完整个体的能力，但其可以分化为个体的所有细胞类型，进而形成身体的所有组织和器官。因此，多能干细胞在组织分化的研究、药物试验及再生医疗等各种领域中被广泛使用，特别是在诱导性多能干细胞(iPSC)建立以后，该领域的研究取得了显著的发展。
在多能干细胞的应用过程中，首先需要在维持多能干细胞的未分化的状态下进行培养，然后对多能干细胞进行诱导分化，以培养得到目标细胞。
在筛选多能干细胞分化诱导剂过程中，目前采用的筛选体系主要有两种：(1)平面培养分化体系，其缺陷是细胞的大规模扩增受到限制，难以获得足够的细胞数量用于研究，且分化效率低，无法对细胞的生长情况以及分化情况做一个精准评估。(2)大体系悬浮培养体系，其缺陷是体系大，成本高，无法进行大规模药筛。
为了克服现有技术的缺陷，本领域技术人员希望开发一种成本低，且能高效筛选多能干细胞分化诱导剂的体系及方法。
发明内容
本发明的目的在于提供一种多能干细胞的培养方法，在增殖培养过程中，能维持多能干细胞的良好的干性(stemness)，在分化过程中，分化效率高、实验间差异小，而且可以高效地筛选多能干细胞的分化诱导剂，对分化情况进行精准评估。本发明提供的多能干细胞的培养方法克服了现有技术中的缺点：现有筛选体系中平面培养分化体系的分化效率低、无法对 细胞的生长情况以及分化情况做精准评估，大体系悬浮培养体系成本高、无法进行大规模药筛等。
为此，第一方面，本发明提供一种多能干细胞的培养方法，采用48孔板为培养容器，细胞接种密度为4.5～6.5×10 5细胞/mL，培养体积为0.4～0.6mL，采用摇床转速为170～190rpm进行培养。
本领域技术人员知晓，本发明所述的48孔板也称48孔培养板、48孔细胞培养板等，每孔容积约为1.7mL，可选用市售的常见的48孔板，例如Thermo Scientific Nunclon所售的48孔板(货号：150687)，Corning所售的48孔板(货号：3548)等。
关于培养容器，多能干细胞的培养往往采用平面培养或大体系悬浮培养，鲜有采用多孔板进行培养的报道；而且，由于多能干细胞本身的特性，多能干细胞的培养无法直接套用其他已分化细胞的培养体系或方法，例如现有技术中已可以根据一些通用性指导规则利用24孔板、48孔板或96孔板等对部分已分化细胞进行培养，而这些培养方法并不适用于多能干细胞的培养。本发明在研究过程中分别使用96孔板、48孔板进行培养，并同步探索相关的接种密度、培养体积、摇床转速等条件。经实验发现，当采用96孔板时，由于培养体积较小，即使采用市售商业摇床的最高转速，仍无法进行悬浮培养，仅能采取静置培养，且培养效果不佳；当采用48孔板时，对培养条件的要求极为苛刻，必须配合特定的细胞接种密度、培养体积和摇床转速，才能获得较佳的培养效果。
在一些实施方式中，所述细胞接种密度约为4.5×10 5细胞/mL、5.5×10 5细胞/mL、6.5×10 5细胞/mL等。
在优选的实施方式中，所述细胞的接种密度为5.5×10 5细胞/mL。
在一些实施方式中，所述培养体系为400μL、500μL、600μL等。
在一些实施方式中，所述摇床转速为170rpm、180rpm、190rpm等。
在培养过程中，培养容器、细胞接种密度、培养体积和摇床转速的配合，对细胞的培养情况会产生综合性影响。首先，当采用本发明所述的48孔板，配合培养体积为0.4～0.6mL、摇床转速为170～190rpm时，可以保证各孔之间的培养基不会发生交叉污染；如果相应地改变培养体积和摇床转速，例如减少培养体积同时提高摇床转速，或者增加培养体积同时降低摇床转速，仍可以保持各孔之间不会发生交叉污染，然而，在偏离了本发明保护范围的这两种情况下，对细胞的聚集形态、细胞粒径的均一性均会造成负面影响，在严重的情况下甚至使培养后的细胞无法维持干性。
本发明在研究过程中发现，当应用48孔板进行培养时，多能干细胞的部分生长情况基本符合本领域技术人员的通常认识，例如，提高细胞接种密度、提高摇床转速有利于细胞快速 生长，增大培养体积、适当提高转速有利于细胞悬浮等。然而，对于多能干细胞的形态(例如聚集体形态、细胞粒径均一性等)和质量(例如干性维持情况等)，接种密度、培养体积和摇床转速对其的影响较大且无明显的规律可循。当培养体系中的一种参数或多种参数偏离本发明所述的48孔板、接种密度为4.5～6.5×10 5细胞/mL、培养体积为400～600μL、摇床转速为170～190rpm时，会对多能干细胞的形态和/或质量造成显著负面影响。
在一些实施方式中，本发明所述多能干细胞的培养方法采用以下培养条件：培养温度为36.6±0.5℃，相对湿度为90±5％，CO 2浓度为5％(v/v)。
在另一些实施方式中，在本发明所述的多能干细胞培养方法中，每20～28h更换新鲜的培养基；优选为每24h更换新鲜的培养基。
在再一些的实施方式中，根据本发明所述的多能干细胞培养方法，包括以下步骤：
(S1)采用48孔板为培养容器，细胞接种密度为4.5～6.5×10 5细胞/mL，培养体积为400～600μL，采用摇床转速为170～190rpm进行培养，得到细胞团悬液；
(S2)取步骤(S1)培养得到的细胞团悬液，制备为单细胞悬液；
(S3)取步骤(S2)制备得到的单细胞悬液，至少重复一次步骤(S1)和(S2)进行培养。
在一些实施方式中，步骤(S1)中，培养1～5d。具体的时间可根据所培养的细胞种类进行确定，例如培养1d、2d、3d、4d或5d等。
在一些实施方式中，步骤(S2)包括：取步骤(S1)培养得到的细胞团悬液，依次进行富集细胞团、消化、过滤、离心、重悬和可选的细胞计数，即制备得到所述单细胞悬液。
在一些实施方式中，步骤(S3)中，所述重复次数为选自1～15的整数。本领域技术人员知晓，所述重复次数即为传代培养的代数，可以根据所培养的细胞种类及生长、分化情况进行确定，例如重复步骤(S1)和(S2)的次数为1次、2次、3次、4次、5次、6次、7次、8次、9次、10次、11次、12次、13次、14次或15次等。
根据本发明所述的多能干细胞的培养方法，所述多能干细胞为胚胎干细胞或诱导性多能干细胞(iPS)。
在一些实施方式中，所述胚胎干细胞为人胚胎干细胞。
本发明的第二方面，提供了所述多能干细胞的培养方法在A1)、A2)、A3)任一方面的应用：
A1)扩增所述多能干细胞；
A2)诱导分化所述多能干细胞；
A3)筛选用于诱导分化所述多能干细胞的诱导剂。
与现有技术相比，本发明的技术方案具有以下显著的进步：
本发明克服了现有技术的偏见，首次采用48孔板对多能干细胞进行培养，从无到有建立了一种适于多能干细胞生长和分化的48孔板悬浮培养体系，且该培养体系可高效地筛选多能干细胞的分化诱导剂。
本发明提供的多能干细胞的培养体系和方法克服了现有技术中平面培养分化体系的分化效率低、无法对细胞的生长情况以及分化情况做精准评估，大体系悬浮培养体系的成本高、无法进行大规模药筛等不足。本发明的技术方案具有以下显著的优势：(1)根据本发明培养的多能干细胞可以维持较好的细胞形态及稳定的增殖倍数，以得到较多的细胞数量用于研究；(2)培养几代后的多能干细胞仍能维持核型正常和较好的细胞表型；(3)培养的多能干细胞在分化过程中细胞团的大小和形状基本保持一致，减少实验间差异；(4)可用此体系进行高通量药物筛选，避免使用价格昂贵的高通量分析仪器，从而显著节省成本。
附图说明
通过阅读下文优选实施方式的详细描述，各种其他的优点和益处对于本领域普通技术人员将变得清楚明了。附图仅用于示出优选实施方式的目的，而并不认为是对本发明的限制。在附图中：
图1：按照本发明的某一培养方法(接种密度5.5×10 5细胞/mL，培养体系600μL，转速180rpm)对人胚胎干细胞H9进行培养，细胞的聚集形态检测结果；
图2：按照本发明的某一培养方法(接种密度5.5×10 5细胞/mL，培养体系600μL，转速180rpm)对人胚胎干细胞H9进行培养，免疫荧光检测结果；
图3：按照本发明的某一培养方法(接种密度5.5×10 5细胞/mL，培养体系600μL，转速180rpm)对人胚胎干细胞H9进行培养，流式细胞术检测结果；
图4：按照本发明的某一培养方法(接种密度5.5×10 5细胞/mL，培养体系600μL，转速180rpm)对人胚胎干细胞H9进行培养，细胞的核型检测结果；
图5：按照本发明的某一培养方法(接种密度5.5×10 5细胞/mL，培养体系600μL，转速180rpm)对诱导性多能干细胞(iPS)进行培养，细胞的聚集形态检测结果；
图6：按照本发明的某一培养方法(接种密度5.5×10 5细胞/mL，培养体系600μL，转速180rpm)对诱导性多能干细胞(iPS)进行培养，流式细胞术检测结果；
图7：按照本发明的某一培养方法(接种密度5.5×10 5细胞/mL，培养体系600μL，转速180rpm)对诱导性多能干细胞(iPS)进行培养，细胞的核型检测结果；
图8：按照本发明的某一培养方法(接种密度4.5×10 5细胞/mL，培养体系400μL，转速180rpm)对人胚胎干细胞H9进行培养，细胞的聚集形态检测结果；
图9：按照本发明的某一培养方法(接种密度4.5×10 5细胞/mL，培养体系500μL，转速180rpm)对人胚胎干细胞H9进行培养，细胞的聚集形态检测结果；
图10：按照本发明的某一培养方法(接种密度4.5×10 5细胞/mL，培养体系600μL，转速180rpm)对人胚胎干细胞H9进行培养，细胞的聚集形态检测结果；
图11：按照本发明的某一培养方法(接种密度6.5×10 5细胞/mL，培养体系600μL，转速180rpm)对人胚胎干细胞H9进行培养，细胞的聚集形态检测结果；
图12：按照本发明的某一培养方法(接种密度5.5×10 5细胞/mL，培养体系600μL，转速170rpm)对人胚胎干细胞H9进行培养，细胞的聚集形态检测结果；
图13：按照本发明的某一培养方法(接种密度5.5×10 5细胞/mL，培养体系600μL，转速190rpm)对人胚胎干细胞H9进行培养，细胞的聚集形态检测结果；
图14：用本发明所述的培养方法培养和诱导分化人胚胎干细胞H9，SANT1类似化学物库后期筛选的分析结果；
图15：用本发明所述的培养方法培养和诱导分化诱导性多能干细胞(iPS)，LDN1931189类似化学物库后期筛选的分析结果；
图16：采用不同接种密度对人胚胎干细胞H9进行96孔板培养，细胞的聚集形态检测结果；
图17：采用不同接种密度对人胚胎干细胞H9进行96孔板培养，免疫荧光检测结果；
图18：采用不同接种密度对人胚胎干细胞H9进行96孔板培养，流式细胞术检测结果；
图19：采用不同接种密度对人胚胎干细胞H9进行96孔板培养，细胞的核型检测结果；
图20：按照对比例的某一培养方法(接种密度4.5×10 5细胞/mL，培养体系700μL，转速180rpm)对人胚胎干细胞H9进行培养，细胞的聚集形态检测结果；
图21：按照对比例的某一培养方法(接种密度3.5×10 5细胞/mL，培养体系600μL，转速180rpm)对人胚胎干细胞H9进行培养，细胞的聚集形态检测结果；
图22：按照对比例的某一培养方法(接种密度5.5×10 5细胞/mL，培养体系600μL，转速160rpm)对人胚胎干细胞H9进行培养，细胞的聚集形态检测结果。
具体实施方式
下面将参照附图更详细地描述本公开的示例性实施方式。虽然附图中显示了本公开的示例性实施方式，然而应当理解，可以以各种形式实现本公开而不应被这里阐述的实施方式所限制。相反，提供这些实施方式是为了能够更透彻地理解本公开，并且能够将本公开的范围完整的传达给本领域的技术人员。
48孔板：Thermo Scientific Nunclon(货号：150687)
96孔板：Thermo Scientific Nunclon(货号：167008)
mTeSR1完全培养基：mTeSR 1 Basal Medium、5×mTeSR 1 Supplement
DE培养基：MCDB131 Basal Medium、1000×Act A、1000x CHIR99021
PGT培养基：MCDB131 Basal Medium、2000×KGF
PP1分化培养基：MCDB131 Basal Medium、2000×KGF、10000×RA、10000×SANT1、10000×LDN
KSR培养基：15％KSR、KO DMEM、L-glutamine、100×non-essential amino acids(NEAA)、1000×β-mercaptoethano
分化培养基：mTeSR with the activin/TGF-b inhibitor SB431542(10mM)and the BMP inhibitor LDN193189
NIM培养基：DMEM/F12、100×N2sup-Plement、50×B27supplement、100×Glutamax、100×NEAA(Gibco)、0.2Mm ascorbic acid
实施例1
本实施例对人胚胎干细胞H9进行动态悬浮培养，并对培养得到的细胞进行相关检测。
多能干细胞动态悬浮培养：
(1)以5.5×10 5细胞/mL的接种密度将人胚胎干细胞H9接种于48孔板，在轨道摇床进行动态悬浮培养，培养基为mTeSR1完全培养基，培养体系为600μL，转速为180rpm，温度37℃，相对湿度90±5％，5％CO 2(v/v)；每培养24h更换新鲜培养基，共培养4d，细胞团粒径为300～400μm，其细胞聚集形态见图1所示。
(2)取出细胞团悬液，经37μm可逆滤器富集细胞团(去除单细胞)，用16mL的Accutase(StemCell)将细胞团反向冲至离心管后置于37℃水浴锅消化15min，期间每间隔5min吹打一次，至出现细胞絮状后加入2倍体积的mTeSR-1培养基终止消化，轻柔混匀后将细胞悬液通过100um筛网过滤(去除细胞絮状)，滤液经离心机(Beckman)RT，200g，4min离心，拨散细胞沉淀，加入含Y-27632的mTeSR-1完全培养基重悬，取适量的细胞悬液通过细胞计数仪(Countstar)进行细胞计数。
(3)取经步骤(2)计数的细胞悬液，重复步骤(1)(2)所述的步骤进行传代培养，共培养4代。
免疫荧光检测：
收集步骤(1)培养得到的细胞团于1.5mL EP管中，PBS润洗3次，加入1mL 4％甲醛固定液室温固定1h，经由低浓度到高浓度的乙醇脱水，1mL二甲苯室温孵育5min，重复3次进行透明并晾干；转移细胞团于石蜡中浸蜡包埋，并对包埋好的样品进行修片、切片和捞 片，将得到的切片于42℃烘箱中烘烤过夜，室温下用二甲苯将切片浸泡5min，重复3次进行脱蜡；95％乙醇浸泡切片2min，70％乙醇浸泡切片2min进行复水，将复水后的切片放于100℃的0.1M EDTA(pH 9.0)的抗原修复液中继续加热20min，加热后将切片快速置于冰中冷却10min，200μL的PBS清洗3次后加入200μL的透化液(PBS+0.1％Triton X-100)室温孵育15min，200μL的PBS清洗3次，加入200μL封闭液(PBS+10％Goat Serum)室温孵育1h，加入一抗4℃孵育过夜，加入二抗室温孵育40min，滴加10μL DAPI(Vector，H1200)至完全覆盖细胞表面后，室温孵育10min，用指甲油对切片进行封片处理；使用倒置荧光显微镜进行观察拍照分析，免疫荧光检测结果见图2所示。根据图2，经本发明所述的方法进行培养后，细胞表达Oct4/SSEA4正常，表明维持了很好的干性(stemness)。
流式细胞术分析：
收集步骤(1)培养得到的细胞团，并制备为单细胞悬液，取3×10 6细胞于EP管中，PBS洗涤2遍，加入1mL Fixation Buffer(BD)固定20min，1×Perm/Wash Buffer(BD)洗涤两遍后，平均分成3份，分别作为unstain组、isotype组和stain组。其中，stain组加入20μL、anti-Oct3/4、anti-SSEA-1、anti-SSEA-4；isotype组加入20μL PerCP-Cy5.5Mouse IgG1、PE Mouse IgM、Alexa
Figure PCTCN2021120868-appb-000001
647 Mouse IgG3；unstain不做处理。室温避光孵育30min，1×Perm/Wash Buffer(BD)洗涤2遍，200μL Stain Buffer重悬，使用BD FACSVerse进行检测，检测结果见图3所示。
核型检测：
收集经培养4代后细胞团，并制备为单细胞悬液，按2×10 5细胞/cm 2接种至T25细胞培养瓶中，待细胞生长达对数生长期转交给第三方机构做核型检测，核型检测报告见图4所示。根据图4所示检测报告，经培养4代后细胞的核型正常。
实施例2
本实施例对诱导性多能干细胞(iPS)进行动态悬浮培养，并对培养得到的细胞进行相关检测。除悬浮培养中的步骤(1)外，其他步骤及检测方法同实施例1。本实施例悬浮培养中的步骤(1)为：
以5.5×10 5细胞/mL的接种密度将诱导性多能干细胞(iPS)接种于48孔板，在轨道摇床进行动态悬浮培养，培养基为mTeSR1完全培养基，培养体系为600μL，转速为180rpm，温度37℃，相对湿度90±5％，5％CO 2(v/v)；每培养24h更换新鲜培养基，共培养4d。
经步骤(1)培养得到的细胞的聚集形态见图5所示；流式检测结果见图6所示，根据图6，经本发明所述的方法进行培养后，细胞表达Oct4/SSEA4正常，表明维持了很好的干性。经培养4代后，细胞的核型检测报告见图7所示，根据图7所示检测报告，经培养4代后细 胞的核型正常。
实施例3
本实施例对人胚胎干细胞H9进行动态悬浮培养，并对培养得到的细胞进行相关检测：
以4.5×10 5细胞/mL的接种密度将人胚胎干细胞H9接种于48孔板，在轨道摇床进行动态悬浮培养，培养基为mTeSR1完全培养基，培养体系为400μL，转速为180rpm，温度37℃，相对湿度90±5％，5％CO 2(v/v)；每培养24h更换新鲜培养基，共培养4d。
培养得到的细胞的聚集形态见图8所示，可以看出细胞聚集体的大小均一，形态较为圆润。
实施例4
本实施例对人胚胎干细胞H9进行动态悬浮培养，并对培养得到的细胞进行相关检测：
以4.5×10 5细胞/mL的接种密度将人胚胎干细胞H9接种于48孔板，在轨道摇床进行动态悬浮培养，培养基为mTeSR1完全培养基，培养体系为500μL，转速为180rpm，温度37℃，相对湿度90±5％，5％CO 2(v/v)；每培养24h更换新鲜培养基，共培养4d。
培养得到的细胞的聚集形态见图9所示，可以看出细胞聚集体的大小均一，形态较为圆润。
实施例5
以4.5×10 5细胞/mL的接种密度将人胚胎干细胞H9接种于48孔板，在轨道摇床进行动态悬浮培养，培养基为mTeSR1完全培养基，培养体系为600μL，转速为180rpm，温度37℃，相对湿度90±5％，5％CO 2(v/v)；每培养24h更换新鲜培养基，共培养4d。
培养得到的细胞的聚集形态见图10所示，可以看出细胞聚集体的大小均一，形态圆润。
实施例6
以6.5×10 5细胞/mL的接种密度将人胚胎干细胞H9接种于48孔板，在轨道摇床进行动态悬浮培养，培养基为mTeSR1完全培养基，培养体系为600μL，转速为180rpm，温度37℃，相对湿度90±5％，5％CO 2(v/v)；每培养24h更换新鲜培养基，共培养4d。
培养得到的细胞的聚集形态见图11所示，可以看出细胞聚集体的大小较为均一，形态圆润。
实施例7
以5.5×10 5细胞/mL的接种密度将人胚胎干细胞H9接种于48孔板，在轨道摇床进行动态悬浮培养，培养基为mTeSR1完全培养基，培养体系为600μL，转速为170rpm，温度37℃，相对湿度90±5％，5％CO 2(v/v)；每培养24h更换新鲜培养基，共培养4d。
培养得到的细胞的聚集形态见图12所示，可以看出细胞聚集体的大小较为均一，形态圆 润。
实施例8
以5.5×10 5细胞/mL的接种密度将人胚胎干细胞H9接种于48孔板，在轨道摇床进行动态悬浮培养，培养基为mTeSR1完全培养基，培养体系为600μL，转速为190rpm，温度37℃，相对湿度90±5％，5％CO 2(v/v)；每培养24h更换新鲜培养基，共培养4d。
培养得到的细胞的聚集形态见图13所示，可以看出出现了个别较大的细胞聚集体，但大多数细胞聚集体仍具有较好的均一性，且形态较为圆润。
实施例9
本实施例将人胚胎干细胞H9诱导分化为胰腺祖细胞，并进行诱导剂筛选。
多能干细胞培养与诱导分化：
以5.5×10 5细胞/mL的接种密度将人胚胎干细胞H9接种于48孔板，在轨道摇床进行动态悬浮培养，培养基为mTeSR1完全培养基，培养体系为600μL，转速为180rpm，温度37℃，相对湿度90±5％，5％CO 2(v/v)；每培养24h更换新鲜培养基，共培养3d。然后更换培养基继续培养，具体为：将培养基更换为DE培养基(S1)培养3d，然后将培养基更换为PGT培养基(S2)培养2d，然后将培养基更换为PP1分化培养基(S3)继续培养。
SANT1类似化学物库筛选：
前期筛选：在上述培养过程中，在更换为PP1分化培养基(S3)时分组建库，各组分别加入10μM的不同的SANT1类似化学物，同时以加入0.25μM SANT1的组作为阳性对照组。在用PP1分化培养基(S3)培养2d后，通过贝克曼流式细胞仪对各组细胞进行检测，分析PDX1的表达。前期命中筛选物如表1所示。
表1
化学物名称 货号 厂家 浓度
Sonidegib T1926 陶素科技 10μM
MK-4101 T6891 陶素科技 10μM
Erismodegib diphosphate T15727 陶素科技 10μM
ALLO-2 T14188 陶素科技 10μM
环巴胺 4449-51-8 南京春秋生物 10μM
后期筛选：
将表1的前期筛选的命中候选化学物分别按0μM、1μM、5μM、10μM、50μM的工作液浓度加入到PP1分化培养基(S3)中，用所得到的培养基对原肠管细胞进行培养，分化到第3天时，将48孔板的细胞转移到96孔板固定并分析PDX1的表达，分析结果如图 14所示。根据图14的分析结果，5μM的环巴胺具有较好的替代效果，可作为将人胚胎干细胞H9诱导分化为胰腺祖细胞的诱导剂。
实施例10
本实施例将诱导性多能干细胞(iPS)诱导分化为神经祖细胞，并进行诱导剂筛选。
多能干细胞培养与诱导分化：
以5.5×10 5细胞/mL的接种密度将诱导性多能干细胞(iPS)接种于48孔板，在轨道摇床进行动态悬浮培养，培养基为mTeSR1完全培养基，培养体系为600μL，转速为180rpm，温度37℃，相对湿度90±5％，5％CO 2(v/v)；每培养24h更换新鲜培养基，共培养3d。然后更换培养基继续培养，具体为：将培养基更换为分化培养基(S1)培养6d，然后将培养基更换为KSR培养基(S2)培养3d，然后将培养基更换为NIM培养基(S3)培养5d。
LDN1931189类似化学物库筛选：
前期：在上述培养过程中，在更换为NIM培养基(S3)时分组建库，各组分别加入10μM的不同的LDN1931189类似化学物，同时以加入5.0μM LDN1931189的组作为阳性对照组。在用NIM培养基(S3)培养5d后，通过贝克曼流式细胞仪对各组细胞进行检测，分析OTX1/2的表达。前期命中筛选物如表2所示。
表2
化学物名称 货号 厂家 浓度
DMH-1 T1942 陶素科技 10μM
SB431542 T1726 陶素科技 10μM
Dorsomorphin T1977 陶素科技 10μM
K02288 T1914 陶素科技 10μM
PD-161570 T23127 陶素科技 10μM
后期：将表2的前期筛选的命中候选化学物分别按0μM、1μM、5μM、10μM、50μM的工作液浓度加入到NIM培养基(S3)中，用所得到的培养基对皮质祖细胞进行培养，分化至第6天时，将48孔板的细胞转移到96孔板固定并分析OTX1/2的表达，分析结果如图15所示。根据图15的分析结果，10μM的DMH-1具有较好的替代效果，可作为将诱导性多能干细胞(iPS)诱导分化为神经细胞的诱导剂。
对比例1
本对比例对人胚胎干细胞H9于96孔板进行静置培养，并对培养得到的细胞进行相关检测。除用以下步骤(1)替换实施例1中所述的步骤(1)外，其他步骤及检测方法同实施例1：
(1)取密度为1×10 5细胞/mL的人胚胎干细胞H9细胞悬液(通过将600μL密度为3.33×10 5细胞/mL的细胞悬液加1400μL培养基制备得到)，按照表3中的培养体系，将其接种于96孔板，按照以下培养条件进行静置培养：培养基为mTeSR1完全培养基，温度37℃，相对湿度90±5％，5％CO 2(v/v)；每培养24h更换新鲜培养基，共培养4d。
表3
Figure PCTCN2021120868-appb-000002
经步骤(1)培养得到的细胞的聚集形态见图16所示，发现粒径出现明显的差异；综合比较后，选择细胞数为3000细胞数/孔的组进行后续免疫荧光检测、流式检测和核型检测。免疫荧光检测的结果如图17所示，可见该体系培养的细胞SSEA4表达不明显，未能很好地维持多能干细胞的干性。流式检测结果见图18所示，根据图18，Oct4/SSEA4阳性细胞群出现拖尾、分群现象，表明培养得到的细胞不纯，有其他细胞。经培养4代后，细胞的核型检测报告见图19所示，根据图19所示检测报告，经培养4代后细胞的核型异常。
对比例2
以4.5×10 5细胞/mL的接种密度将人胚胎干细胞H9接种于48孔板，在轨道摇床进行动态悬浮培养，培养基为mTeSR1完全培养基，培养体系为700μL，转速为180rpm，温度37℃，相对湿度90±5％，5％CO 2(v/v)；每培养24h更换新鲜培养基，共培养4d。
培养得到的细胞的聚集形态见图20所示，可以看出当培养体积增大为700μL时，细胞聚集体大小不一，有明显较大的细胞团出现。
对比例3
以3.5×10 5细胞/mL的接种密度将人胚胎干细胞H9接种于48孔板，在轨道摇床进行动态悬浮培养，培养基为mTeSR1完全培养基，培养体系为600μL，转速为180rpm，温度37℃，相对湿度90±5％，5％CO 2(v/v)；每培养24h更换新鲜培养基，共培养4d。
培养得到的细胞的聚集形态见图21所示，可以看出细胞聚集体的大小均一性非常差，且有融合现象出现。
对比例4
以5.5×10 5细胞/mL的接种密度将人胚胎干细胞H9接种于48孔板，在轨道摇床进行动态悬浮培养，培养基为mTeSR1完全培养基，培养体系为600μL，转速为160rpm，温度 37℃，相对湿度90±5％，5％CO 2(v/v)；每培养24h更换新鲜培养基，共培养4d。
培养得到的细胞的聚集形态见图22所示，可以看出细胞数较少，不能很好地成团，且细胞团粒径大小不均一。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。

Claims (9)

  1. 一种多能干细胞的培养方法，其特征在于，采用48孔板为培养容器，细胞接种密度为4.5～6.5×10 5细胞/mL，培养体积为0.4～0.6mL，采用摇床转速为170～190rpm进行培养。
  2. 如权利要求1所述的多能干细胞的培养方法，其特征在于，培养温度为36.5±0.5℃，相对湿度为90±5％，CO 2浓度为5％(v/v)。
  3. 如权利要求1所述的多能干细胞的培养方法，其特征在于，每20～28h更换新鲜的培养基。
  4. 如权利要求1所述的多能干细胞的培养方法，其特征在于，所述多能干细胞为胚胎干细胞或诱导性多能干细胞。
  5. 如权利要求1～4任一项所述的多能干细胞的培养方法，其特征在于，包括以下步骤：
    (S1)采用48孔板为培养容器，细胞接种密度为4.5～6.5×10 5细胞/mL，培养体积为0.4～0.6mL，采用摇床转速为170～190rpm进行培养，得到细胞团悬液；
    (S2)取步骤(S1)培养得到的细胞团悬液，制备为单细胞悬液；
    (S3)取步骤(S2)制备得到的单细胞悬液，至少重复一次步骤(S1)和(S2)进行培养。
  6. 如权利要求5所述的多能干细胞的培养方法，其特征在于，步骤(S2)包括：取步骤(S1)培养得到的细胞团悬液，依次进行富集细胞团、消化、过滤、离心、重悬和可选的细胞计数，即制备得到所述单细胞悬液。
  7. 如权利要求5所述的多能干细胞的培养方法，其特征在于，步骤(S1)中，培养时间为1～5d。
  8. 如权利要求5所述的多能干细胞的培养方法，其特征在于，步骤(S3)中，所述重复次数为选自1～15的整数。
  9. 权利要求1～8任一项所述的多能干细胞的培养方法在A1)、A2)、A3)任一方面的应用：
    A1)扩增所述多能干细胞；
    A2)诱导分化所述多能干细胞；
    A3)筛选用于诱导分化所述多能干细胞的诱导剂。
PCT/CN2021/120868 2021-03-12 2021-09-27 一种多能干细胞的培养方法 WO2022188395A1 (zh)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104284977A (zh) * 2012-05-07 2015-01-14 詹森生物科技公司 人胚胎干细胞向胰腺内胚层的分化
CN112048470A (zh) * 2020-09-17 2020-12-08 深圳丹伦基因科技有限公司 一种利用人诱导多能干细胞制备临床级别间充质干细胞制剂的方法
US20210062150A1 (en) * 2019-09-03 2021-03-04 Allogene Therapeutics, Inc. Methods of preparing t cells for t cell therapy
CN113046306A (zh) * 2021-03-12 2021-06-29 广东东阳光药业有限公司 一种多能干细胞的培养方法

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014170549A1 (en) * 2013-04-16 2014-10-23 Glykos Finland Oy A method for generating induced pluripotent stem cells
CN104342439B (zh) * 2013-07-23 2017-06-23 中国科学院遗传与发育生物学研究所 miR‑7及其应用
AU2015313264B2 (en) * 2014-09-08 2021-11-04 Riken Method for producing cerebellar progenitor tissue
CN104725511B (zh) * 2015-02-16 2018-01-12 华南农业大学 一种猪肠道干细胞的分离培养方法
US20190010452A1 (en) * 2016-01-22 2019-01-10 National University Corporation Nagoya University Differentiation induction from human pluripotent stem cells into hypothalamic neurons
CN109679893A (zh) * 2019-01-17 2019-04-26 浙江工商大学 基于小鼠肠道干细胞的肠上皮单层培养及表征方法
CN110055214B (zh) * 2019-05-02 2022-09-30 南华生物医药股份有限公司 一种人参皂苷促进脐带间充质干细胞体外增殖的用途

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104284977A (zh) * 2012-05-07 2015-01-14 詹森生物科技公司 人胚胎干细胞向胰腺内胚层的分化
US20210062150A1 (en) * 2019-09-03 2021-03-04 Allogene Therapeutics, Inc. Methods of preparing t cells for t cell therapy
CN112048470A (zh) * 2020-09-17 2020-12-08 深圳丹伦基因科技有限公司 一种利用人诱导多能干细胞制备临床级别间充质干细胞制剂的方法
CN113046306A (zh) * 2021-03-12 2021-06-29 广东东阳光药业有限公司 一种多能干细胞的培养方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LACO FILIP, LAM ALAN TIN-LUN, WOO TSUNG-LIANG, TONG GERINE, HO VALERIE, SOONG POH-LOONG, GRISHINA ELINA, LIN KUN-HAN, REUVENY SHAU: "Selection of human induced pluripotent stem cells lines optimization of cardiomyocytes differentiation in an integrated suspension microcarrier bioreactor", STEM CELL RESEARCH & THERAPY, vol. 11, no. 1, 1 December 2020 (2020-12-01), XP055966544, DOI: 10.1186/s13287-020-01618-6 *
QI, KAI: "Optimized Study on Isolation and Cultivation of Human Umbilical Cord Mesenchymal Stem Cells", CHINA DOCTORAL DISSERTATIONS AND MASTER’S THESES FULL-TEXT DATABASE (MASTER), MEDICINE AND HEALTH SCIENCES, 18 May 2011 (2011-05-18), XP055966539, [retrieved on 20220929] *

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