TW202235612A - Method for culturing pluripotent stem cells - Google Patents

Abstract

The invention relates to a method for culturing pluripotent stem cells. The method adopts a 48-pore plate as a culture container, the cell inoculation density is 4.5-6.5*105 cells/mL, the culture volume is 0.4-0.6 mL, and the rotating speed of a shaking table is 170-190 rpm. According to the method, a 48-pore plate suspension culture system suitable for growth and differentiation of the pluripotent stem cells is established from nothing to thing, and the culture system can be used for efficiently screening a differentiation inducer of the pluripotent stem cells. The culture method provided by the invention has the advantages of low cost, high efficiency, capability of maintaining better cellular morphology, stable proliferation multiple, capability of maintaining better karyotype and phenotype, smaller difference between experiments in the differentiation process and the like.

Description

Methods of culturing pluripotent stem cells

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 state, in vitro proliferation and differentiation potential, etc. 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 have lost the ability to develop into a full individual, but they can differentiate into all of an individual's cell types, which in turn form all of the body's tissues and organs. 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 (iPSC), research in this field has achieved remarkable development.

In the application process of pluripotent stem cells, it is first necessary to maintain the undifferentiated state of pluripotent stem cells for culture, and then induce differentiation of pluripotent stem cells to obtain target cells.

In the process of screening pluripotent stem cell differentiation inducers, there are two main screening systems currently used: (1) planar culture differentiation system, its defect is 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, it is impossible to make an accurate assessment of the growth and differentiation of the cells. (2) The large system suspension culture system has the disadvantages of large system, high cost, and large-scale drug screening cannot be carried out.

In order to overcome the defects of the prior art, those skilled in the art hope to develop a low-cost and efficient screening system and method for pluripotent stem cell differentiation inducers.

The object of the present invention is to provide a method for culturing pluripotent stem cells, which can maintain good stemness (stemness) of pluripotent stem cells during the proliferation culture process, and has high differentiation efficiency and small differences between experiments during the differentiation process, and can Efficiently screen for differentiation inducers of pluripotent stem cells, and accurately evaluate the differentiation status. The method for cultivating pluripotent stem cells provided by the present invention overcomes the shortcomings of the prior art: the differentiation efficiency of the planar culture differentiation system in the existing screening system is low, the growth and differentiation of cells cannot be accurately evaluated, and the cost of the large-system suspension culture system is low. High, unable to carry out large-scale drug screening, etc.

Therefore, in the first aspect, the present invention provides a method for culturing pluripotent stem cells. A 48-well plate is used as a culture container, the cell seeding density is 4.5-6.5×10 ⁵ cells/mL, and the culture volume is 0.4-0.6 mL. The bed rotation speed was 170~190 rpm for cultivation.

Those skilled in the art know that the 48-well plate of 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. Commercially available common 48-well plates can be selected, such as Thermo Scientific 48-well plates sold by Nunclon (Cat. No.: 150687), 48-well plates sold by Corning (Cat. No.: 3548), etc.

As for the culture container, the culture of pluripotent stem cells is often carried out by plane culture or large-system suspension culture, and there are few reports on the culture 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 cultures. Cell culture systems or methods, for example, in the prior art, 24-well plates, 48-well plates, or 96-well plates can be used to culture some differentiated cells according to some general guidelines, but these culture methods are not suitable for pluripotent Stem cell culture. In the research process of the present invention, 96-well plates and 48-well plates are used for culture respectively, and conditions such as inoculation density, culture volume, and shaker speed are simultaneously explored. It has been found through experiments that when a 96-well plate is used, due to the small culture volume, suspension culture cannot be carried out even with the highest rotational speed of a commercially available shaker, and only static culture can be adopted, and the culture effect is not good; When using a well plate, the requirements for the culture conditions are extremely strict, and a specific cell seeding density, culture volume and shaker speed must be matched to obtain a better culture effect.

In some embodiments, the cell seeding density is about 4.5×10 ⁵ cells/mL, 5.5×10 ⁵ cells/mL, 6.5×10 ⁵ cells/mL, etc.

In a preferred embodiment, the seeding density of the cells is 5.5×10 ⁵ cells/mL.

In some embodiments, the culture system is 400 μL, 500 μL, 600 μL, etc.

In some embodiments, the rotating speed of the shaker is 170 rpm, 180 rpm, 190 rpm, etc.

During the culture process, the cooperation of the culture container, cell seeding density, culture volume and shaker speed will have a comprehensive impact on the culture of the cells. First of all, when the 48-well plate of the present invention is used, with a culture volume of 0.4-0.6 mL and a shaking table rotation speed of 170-190 rpm, it can be ensured that no cross-contamination of the culture medium between the wells will occur; if the culture medium is changed accordingly Culture volume and shaker speed, such as reducing the culture volume while increasing the shaker speed, or increasing the culture volume while reducing the shaker speed, can still keep cross-contamination from occurring between each well, however, when deviating from the protection scope of the present invention In both cases, it will have a negative impact on the aggregate shape of the cells and the uniformity of the cell particle size, and in severe cases, the cultured cells may even be unable to maintain stemness.

During the research process, the present invention found that when a 48-well plate was used for culture, the partial growth of pluripotent stem cells was basically in line with the common knowledge of those skilled in the art, for example, increasing the cell seeding density and the speed of the shaker were conducive to the rapid growth of cells , increase the culture volume, increase the rotation speed appropriately to facilitate cell suspension, etc. However, for the morphology of pluripotent stem cells (such as aggregate shape, uniformity of cell particle size, etc.) rules to follow. When one or more parameters in the culture system deviate from the 48-well plate described in the present invention, the seeding density is 4.5-6.5× ¹⁰⁵ cells/mL, the culture volume is 400-600 μL, and the shaker speed is 170-190 rpm , can significantly negatively affect the morphology and/or quality of pluripotent stem cells.

In some embodiments, 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).

In other embodiments, in the method for culturing pluripotent stem cells of the present invention, the fresh medium is replaced every 20-28 h; preferably, the fresh medium is replaced every 24 h.

In some other embodiments, the method for culturing pluripotent stem cells according to the present invention comprises the following steps:

(S1) Use a 48-well plate as the culture container, the cell seeding density is 4.5-6.5×10 ⁵ cells/mL, the culture volume is 400-600 μL, and the shaker speed is 170-190 rpm for culture to obtain a cell mass suspension ;

(S2) taking the cell mass suspension cultured in step (S1) and preparing it as a single cell suspension;

(S3) Taking the single cell suspension prepared in step (S2), repeating steps (S1) and (S2) at least once for culturing.

In some embodiments, in step (S1), culture for 1-5 days. The specific time can be determined according to the type of cells to be cultured, for example, culture for 1 day, 2 days, 3 days, 4 days or 5 days, etc.

In some embodiments, step (S2) includes: taking the cell mass suspension cultured in step (S1), performing successively enrichment of cell mass, digestion, filtration, centrifugation, resuspension and optional cell counting, that is, the prepared The single cell suspension.

In some embodiments, in step (S3), the number of repetitions is an integer selected from 1-15. Those skilled in the art know that the number of repetitions is the number of generations of subculture, which can be determined according to the type of cells cultured and the growth and differentiation conditions. For example, the number of repetitions of steps (S1) and (S2) is 1 time, 2 times Times, 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.

According to the method for culturing pluripotent stem cells of the present invention, the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells (iPS).

In some embodiments, 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):

A1) expanding the pluripotent stem cells;

A2) inducing differentiation of the pluripotent stem cells;

A3) Screening an inducer for inducing differentiation of the pluripotent stem cells.

Compared with the prior art, the technical solution of the present invention has the following remarkable progress:

The present invention overcomes the prejudice of the prior art. For the first time, a 48-well plate is used to cultivate pluripotent stem cells, and a 48-well plate suspension culture system suitable for the growth and differentiation of pluripotent stem cells is established from scratch, and the culture system can be highly efficient. Screening for differentiation inducers of pluripotent stem cells.

The culture system and method of pluripotent stem cells provided by the present invention overcome the low differentiation efficiency of the planar culture differentiation system in the prior art, the inability to accurately evaluate the growth and differentiation of cells, the high cost of the large-system suspension culture system, and the inability to carry out Insufficient large-scale drug screening and so on. 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 factor, so as to obtain a large number of cells for research; (2) culture After several generations, the pluripotent stem cells can still maintain a normal karyotype and a good cell phenotype; (3) The size and shape of the cell clusters of the cultured pluripotent stem cells during the differentiation process are basically consistent, reducing the differences between experiments; (4) This system can be used for high-throughput drug screening, avoiding the use of expensive high-throughput analytical instruments, thereby significantly saving costs.

Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided for more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art.

48-well plate: Thermo Scientific Nunclon (Cat. No. 150687)

96-well plate: Thermo Scientific Nunclon (Cat. No. 167008)

Complete mTeSR1 medium: mTeSR 1 Basal Medium, 5×mTeSR 1 Supplement

DE medium: MCDB131 Basal Medium, 1000×Act 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×β-mercaptoethano

Differentiation medium: mTeSR with the activin/TGF-b inhibitor SB431542 (10 mM) and the BMP inhibitor LDN193189

NIM medium: DMEM/F12, 100×N2 sup-Plement, 50×B27 supplement, 100×Glutamax, 100×NEAA (Gibco), 0.2Mm ascorbic acid

Example 1

In this example, the dynamic suspension culture of human embryonic stem cells H9 was carried out, and relevant detection was carried out on the cultured cells.

Dynamic suspension culture of pluripotent stem cells:

(1) Inoculate human embryonic stem cells H9 in a 48-well plate at a seeding density of 5.5×10 ⁵ cells/mL, and carry out dynamic suspension culture on an orbital shaker. The medium is mTeSR1 complete medium, the culture system is 600 μL, and the rotation speed is 180 rpm , temperature 37 ℃, relative humidity 90±5%, 5% CO ₂ (v/v); fresh medium was replaced every 24 hours of culture, co-cultured for 4 days, the particle size of cell clusters was 300-400 μm, and the cell aggregation morphology was shown in Figure 1 shows.

(2) Take out the cell suspension, pass through a 37 μm reversible filter to enrich the cell cluster (remove single cells), use 16 mL of Accutase (StemCell) to backwash the cell cluster into a centrifuge tube and place it in a 37 °C water bath for 15 min, pipetting at intervals of 5 min during the period, and adding 2 times the volume of mTeSR-1 medium to stop digestion after cell flocculation, and gently mixing, then filtering the cell suspension through a 100 um sieve (to remove cell flocculation), and the filtrate Centrifuge in a centrifuge (Beckman) at RT, 200 g, 4 min to loosen the cell pellet, add mTeSR-1 complete medium containing Y-27632 to resuspend, take an appropriate amount of cell suspension and count the cells by a cell counter (Countstar) .

(3) Take the cell suspension counted in step (2), repeat the steps described in step (1) and (2) for subculture, and co-culture for 4 generations.

Immunofluorescence detection:

Collect the cell mass obtained in step (1) into a 1.5 mL EP tube, rinse with PBS three times, add 1 mL of 4% formaldehyde fixative solution to fix at room temperature for 1 h, dehydrate through low to high concentration of ethanol, and 1 mL of two Incubate in toluene at room temperature for 5 min, repeat 3 times to make transparent and dry; transfer the cell mass to paraffin and embed in wax, and trim, slice and remove the embedded samples, and store the obtained slices at 42 ℃ Baked in an oven overnight, soaked the slices in xylene at room temperature for 5 min, and repeated 3 times for dewaxing; soaked the slices in 95% ethanol for 2 min, soaked the slices in 70% ethanol for 2 min to rehydrate, and rehydrated the slices Place in 0.1 M EDTA (pH 9.0) antigen retrieval solution at 100 ℃ and continue heating for 20 minutes. After heating, quickly place the slices in ice for 10 minutes, wash with 200 μL of PBS for 3 times, and then add 200 μL of permeabilization solution (PBS + 0.1% Triton X-100) incubate at room temperature for 15 min, wash 3 times with 200 μL of PBS, add 200 μL of blocking solution (PBS + 10% Goat Serum), incubate at room temperature for 1 h, add primary antibody and incubate overnight at 4 °C , add secondary antibody and incubate at room temperature for 40 min, drop 10 μL of DAPI (Vector, H1200) to completely cover the cell surface, incubate at room temperature for 10 min, seal the sections with nail polish; observe with an inverted fluorescent microscope Take photos for analysis, and the results of immunofluorescence detection are shown in Figure 2. According to FIG. 2 , after being cultured by the method of the present invention, the cells express Oct4/SSEA4 normally, indicating that a good stemness is maintained.

Flow Cytometry Analysis:

Collect the cell cluster obtained in step (1) and prepare it as a single cell suspension. Take 3×10 ⁶ cells in an EP tube, wash them twice with PBS, add 1 mL Fixation Buffer (BD) for 20 min, 1×Perm /Wash Buffer (BD) was washed twice, and divided into 3 parts on average, which were used as unstain group, isotype group and stain group respectively. Among them, 20 μL, anti-Oct3/4, anti-SSEA-1, anti-SSEA-4 were added to the stain group; 20 μL PerCP-Cy5.5 Mouse IgG1, PE Mouse IgM, Alexa Fluor® 647 Mouse IgG3 were added to the isotype group; The unstain group will not be processed. Incubate at room temperature in the dark for 30 min, wash 2 times with 1×Perm/Wash Buffer (BD), resuspend in 200 μL Stain Buffer, and use BD FACSVerse for detection. The detection results are shown in Figure 3.

Karyotype detection:

Collect cell clusters after 4 generations of culture, prepare single cell suspension, inoculate into T25 cell culture flasks at 2×10 ⁵ cells/cm ² , and transfer to the third-party manufacturer for karyotype detection after 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 after 4 passages was normal.

Example 2

In this example, induced pluripotent stem cells (iPS) were cultured in dynamic suspension, and the cultured cells were tested. Except for the step (1) in the suspension culture, other steps and detection methods are the same as in Example 1. Step (1) in the suspension culture of this embodiment is:

Induced pluripotent stem cells (iPS) were inoculated in 48-well plates at a seeding density of 5.5×10 ⁵ cells/mL, and dynamic suspension culture was carried out on an orbital shaker. The medium was mTeSR1 complete medium, the culture system was 600 μL, and the rotation speed was 180 rpm, temperature 37 ℃, relative humidity 90±5%, 5% CO ₂ (v/v); fresh medium was replaced every 24 h for co-cultivation for 4 days.

The aggregation morphology of the cells cultured in step (1) is shown in Figure 5; the results of flow cytometry are shown in Figure 6. According to Figure 6, after being cultured by the method of the present invention, the cells express Oct4/SSEA4 normally, indicating that Maintained a good dryness. After 4 generations of culture, 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.

Example 3

In this example, the dynamic suspension culture of human embryonic stem cells H9 was carried out, and relevant tests were carried out on the cultured cells:

Human embryonic stem cells H9 were inoculated in a 48-well plate at a seeding density of 4.5×10 ⁵ cells/mL, and cultured in a dynamic suspension on an orbital shaker. ℃, relative humidity 90±5%, 5% CO ₂ (v/v); fresh medium was replaced every 24 h for co-cultivation for 4 days.

The aggregation 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.

Example 4

In this example, the dynamic suspension culture of human embryonic stem cells H9 was carried out, and relevant tests were carried out on the cultured cells:

Human embryonic stem cells H9 were inoculated in a 48-well plate at a seeding density of 4.5×10 ⁵ cells/mL, and cultured in a dynamic suspension on an orbital shaker. ℃, relative humidity 90±5%, 5% CO ₂ (v/v); fresh medium was replaced every 24 h for co-cultivation for 4 days.

The aggregation 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.

Example 5

Human embryonic stem cells H9 were inoculated in 48-well plates at a seeding density of 4.5×10 ⁵ cells/mL, and dynamic suspension culture was carried out on an orbital shaker. ℃, relative humidity 90±5%, 5% CO ₂ (v/v); fresh medium was replaced every 24 h for co-cultivation for 4 days.

The aggregation 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.

Example 6

Human embryonic stem cells H9 were inoculated in a 48-well plate at a seeding density of 6.5×10 ⁵ cells/mL, and dynamic suspension culture was carried out on an orbital shaker. ℃, relative humidity 90±5%, 5% CO ₂ (v/v); fresh medium was replaced every 24 h for co-cultivation for 4 days.

The aggregation 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.

Example 7

Human embryonic stem cells H9 were inoculated in a 48-well plate at a seeding density of 5.5×10 ⁵ cells/mL, and dynamic suspension culture was carried out on an orbital shaker. ℃, relative humidity 90±5%, 5% CO ₂ (v/v); fresh medium was replaced every 24 h for co-cultivation for 4 days.

The aggregation 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.

Example 8

Human embryonic stem cells H9 were inoculated in a 48-well plate at a seeding density of 5.5×10 ⁵ cells/mL, and cultured in a dynamic suspension on an orbital shaker. ℃, relative humidity 90±5%, 5% CO ₂ (v/v); fresh medium was replaced every 24 h for co-cultivation for 4 days.

The aggregation morphology of the cultured cells is shown in Figure 13. It can be seen that individual large cell aggregates appeared, but most of the cell aggregates still had good uniformity and a relatively round shape.

Example 9

In this example, human embryonic stem cells H9 were induced to differentiate into pancreatic progenitor cells, and the induction agent was screened.

Pluripotent stem cell culture and induced differentiation:

Human embryonic stem cells H9 were inoculated in a 48-well plate at a seeding density of 5.5×10 ⁵ cells/mL, and cultured in a dynamic suspension on an orbital shaker. ℃, relative humidity 90±5%, 5% CO ₂ (v/v); fresh medium was replaced every 24 h for co-cultivation for 3 days. Then the medium was replaced to continue the culture, specifically: the medium was changed to DE medium (S1) for 3 days, then the medium was changed to PGT medium (S2) for 2 days, and then the medium was changed to PP1 differentiation medium (S3) to continue the culture.

SANT1 analogue chemical library screening:

Preliminary screening: During the above culture process, when replacing with PP1 differentiation medium (S3), the library was divided into groups, and 10 μM of different SANT1 analogue chemicals were added to each group, and the group added with 0.25 μM SANT1 was used as the positive control group. After being cultured with PP1 differentiation medium (S3) for 2 days, the cells in each group were detected by Beckman flow cytometry to analyze the expression of PDX1. The early hit screens are shown in Table 1. [Table 1] chemical name Item No. factory concentration Sonidegib T1926 Tao Su Technology 10μM MK-4101 T6891 Tao Su Technology 10μM Erismodegib diphosphate T15727 Tao Su Technology 10μM ALLO-2 T14188 Tao Su Technology 10μM cyclopamine 4449-51-8 Nanjing Chunqiu Biology 10μM

Post screening:

The hit candidate chemicals screened in the previous stage in Table 1 were added to the PP1 differentiation medium (S3) at the working concentration of 0 μM, 1 μM, 5 μM, 10 μM, and 50 μM, and the obtained medium was used to treat the original intestinal tube cells After culturing, the cells in the 48-well plate were transferred to a 96-well plate to fix and analyze the expression of PDX1 on the third day of differentiation. The analysis results are shown in FIG. 14 . According to the analysis results in Figure 14, 5 μM cyclopamine has a good substitution effect and can be used as an inducer to induce the differentiation of human embryonic stem cells H9 into pancreatic progenitor cells.

Example 10

In this example, induced pluripotent stem cells (iPS) were induced to differentiate into neural progenitor cells, and the inducer was screened.

Pluripotent stem cell culture and induced differentiation:

Induced pluripotent stem cells (iPS) were inoculated in 48-well plates at a seeding density of 5.5×10 ⁵ cells/mL, and dynamic suspension culture was carried out on an orbital shaker. The medium was mTeSR1 complete medium, the culture system was 600 μL, and the rotation speed was 180 rpm, temperature 37 ℃, relative humidity 90±5%, 5% CO ₂ (v/v); fresh medium was replaced every 24 h, and co-cultured for 3 days. Then the medium was replaced to continue the culture, specifically: the medium was replaced with differentiation medium (S1) for 6 days, then the medium was replaced with KSR medium (S2) for 3 days, and then the medium was replaced with NIM medium (S3) for 5 days.

LDN1931189 similar chemical library screening:

Early stage: During the above culture process, the library was divided into NIM medium (S3), and 10 μM of different LDN1931189 analogues were added to each group, and the group added with 5.0 μM LDN1931189 was used as the positive control group. After being cultured in NIM medium (S3) for 5 days, the cells in each group were detected by Beckman flow cytometry to analyze the expression of OTX1/2. The early hit screens are shown in Table 2. [Table 2] chemical name Item No. factory concentration DMH-1 T1942 Tao Su Technology 10μM SB431542 T1726 Tao Su Technology 10μM Dorsomorphin T1977 Tao Su Technology 10μM K02288 T1914 Tao Su Technology 10μM PD-161570 T23127 Tao Su Technology 10μM

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 μM, 1 μM, 5 μM, 10 μM, and 50 μM, respectively, and the obtained medium was used to treat the cortical progenitors. The cells were cultured, and when they were differentiated to day 6, the cells in the 48-well plate were transferred to a 96-well plate for fixation and the expression of OTX1/2 was analyzed. The analysis results are shown in FIG. 15 . According to the analysis results in Figure 15, 10 μM 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 neurons.

Comparative example 1

In this comparative example, human embryonic stem cells H9 were statically cultured in a 96-well plate, and relevant tests were performed on the cultured cells. Except that the step (1) described in Example 1 is replaced with the following step (1), other steps and detection methods are the same as in Example 1:

(1) Take human embryonic stem cell H9 cell suspension with a density of 1×10 ⁵ cells/mL (prepared by adding 600 μL of cell suspension with a density of 3.33×10 ⁵ cells/mL to 1400 μL of medium), according to Table 3 Inoculate the culture system in 96-well plate, and carry out static culture according to the following culture conditions: the medium is mTeSR1 complete medium, the temperature is 37 ℃, the relative humidity is 90±5%, and 5% CO ₂ (v/v); After 24 h of cultivation, fresh medium was replaced, and co-cultivation was carried out for 4 d. [table 3] group Cells/well Density of cell suspension cell suspension volume Media Supplement Volume 1 500 1×10 ⁵ cells/mL 5uL 195uL 2 1000 10uL 190uL 3 3000 30uL 170uL 4 5000 50uL 150uL

The aggregation morphology of the cells cultured in step (1) is shown in Figure 16, and there are obvious differences in particle size; after a comprehensive comparison, the group with a cell number of 3000 cells/well was selected for subsequent immunofluorescence detection and flow cytometry. detection and karyotyping. The results of immunofluorescence detection are shown in Figure 17. It can be seen that the expression of SSEA4 in cells cultured in this system is not obvious, and the stemness of pluripotent stem cells cannot be well maintained. The results of the flow cytometry test are shown in Figure 18. According to Figure 18, the Oct4/SSEA4 positive cell populations appear tailing and clustering, indicating that the cultured cells are impure and contain other cells. After 4 generations of culture, the karyotype test report of the cells is shown in Figure 19. According to the test report shown in Figure 19, the karyotype of the cells after 4 generations of culture is abnormal.

Comparative example 2

Human embryonic stem cells H9 were inoculated in a 48-well plate at a seeding density of 4.5×10 ⁵ cells/mL, and cultured in a dynamic suspension on an orbital shaker. ℃, relative humidity 90±5%, 5% CO ₂ (v/v); fresh medium was replaced every 24 h for co-cultivation for 4 days.

The aggregation morphology of the cultured cells is shown in Figure 20. It can be seen that when the culture volume was increased to 700 μL, the cell aggregates were of different sizes, and obviously larger cell clusters appeared.

Comparative example 3

Human embryonic stem cells H9 were inoculated in a 48-well plate at a seeding density of 3.5×10 ⁵ cells/mL, and cultured in a dynamic suspension on an orbital shaker. ℃, relative humidity 90±5%, 5% CO ₂ (v/v); fresh medium was replaced every 24 h for co-cultivation for 4 days.

The aggregation 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 fusion phenomenon.

Comparative example 4

Human embryonic stem cells H9 were inoculated in 48-well plates at a seeding density of 5.5×10 ⁵ cells/mL, and dynamic suspension culture was carried out on an orbital shaker. ℃, relative humidity 90±5%, 5% CO ₂ (v/v); fresh medium was replaced every 24 h for co-cultivation for 4 days.

The aggregation morphology of the cultured cells is shown in Figure 22. It can be seen that the number of cells is small, they cannot be well aggregated, and the size of the cell aggregates is not uniform.

The above is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed in the present invention can easily think of changes or Replacement should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the protection scope of the claims.

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiment. The drawings are only for the purpose of illustrating a preferred embodiment and are not to be considered as limiting the invention. In the accompanying drawings: [Fig. 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 μL, rotation speed 180 rpm), and the aggregated morphology of the cells Test results. [Figure 2] Human embryonic stem cell H9 was cultured according to a certain culture method of the present invention (seeding density 5.5×10 ⁵ cells/mL, culture system 600 μL, rotation speed 180 rpm), and the results of immunofluorescence detection. [Figure 3] Human embryonic stem cell H9 was cultured according to a certain culture method of the present invention (seeding density 5.5×10 ⁵ cells/mL, culture system 600 μL, rotation speed 180 rpm), and flow cytometry detection results. [Figure 4] Human embryonic stem cell H9 was cultured according to a certain culture method of the present invention (inoculation density 5.5×10 ⁵ cells/mL, culture system 600 μL, rotation speed 180 rpm), and the karyotype detection results of the cells. [Figure 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 μL, rotation speed 180 rpm), and the aggregation morphology of the cells was detected result. [Figure 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 μL, rotation speed 180 rpm), and flow cytometry detection result. [Figure 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 μL, rotation speed 180 rpm), and the karyotype detection of the cells result. [Fig. 8] Human embryonic stem cell H9 was cultured according to a certain culture method of the present invention (inoculation density 4.5×10 ⁵ cells/mL, culture system 400 μL, rotation speed 180 rpm), and the detection results of cell aggregation morphology. [Fig. 9] Human embryonic stem cell H9 was cultured according to a certain culture method of the present invention (inoculation density 4.5×10 ⁵ cells/mL, culture system 500 μL, rotation speed 180 rpm), and the detection results of cell aggregation morphology. [Fig. 10] Human embryonic stem cell H9 was cultured according to a certain culture method of the present invention (seeding density 4.5×10 ⁵ cells/mL, culture system 600 μL, rotation speed 180 rpm), and the detection results of cell aggregation morphology. [Figure 11] Human embryonic stem cells H9 were cultured according to a certain culture method of the present invention (seeding density 6.5×10 ⁵ cells/mL, culture system 600 μL, rotation speed 180 rpm), and the detection results of cell aggregation morphology. [Fig. 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 μL, rotation speed 170 rpm), and the detection results of cell aggregation morphology. [Figure 13] Human embryonic stem cell H9 was cultured according to a certain culture method of the present invention (inoculation density 5.5×10 ⁵ cells/mL, culture system 600 μL, rotation speed 190 rpm), and the detection results of cell aggregation morphology. [Fig. 14] The human embryonic stem cell H9 was cultured and induced to differentiate by the culture method of the present invention, and the analysis result of the later stage screening of the SANT1 analogue chemical library. [Fig. 15] The culture method of the present invention was used to culture and induce the differentiation of induced pluripotent stem (iPS) cells, and the analysis results of the later screening of LDN1931189 similar 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 inoculation 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 inoculation densities, and the karyotype detection results of the cells. [Figure 20] Human embryonic stem cells H9 were cultured according to a certain culture method of the comparative example (inoculation density 4.5×10 ⁵ cells/mL, culture system 700 μL, rotation speed 180 rpm), and the detection results of cell aggregation morphology. [Figure 21] Human embryonic stem cells H9 were cultured according to a certain culture method of the comparative example (inoculation density 3.5×10 ⁵ cells/mL, culture system 600 μL, rotation speed 180 rpm), and the detection results of cell aggregation morphology. [Figure 22] Human embryonic stem cells H9 were cultured according to a certain culture method of the comparative example (inoculation density 5.5×10 ⁵ cells/mL, culture system 600 μL, rotation speed 160 rpm), and the detection results of cell aggregation morphology.

Claims (9)

A method for culturing pluripotent stem cells, wherein a 48-well plate is used as a culture container, the cell seeding density is 4.5-6.5×10 ⁵ cells/mL, the culture volume is 0.4-0.6 mL, and the rotation speed of a shaker is 170-190 rpm. nourish. The method for culturing pluripotent stem cells as described in Claim 1, wherein the culturing temperature is 36.5±0.5°C, the relative humidity is 90±5%, and the CO ₂ concentration is 5% (v/v). The method for culturing pluripotent stem cells as described in Claim 1, wherein the fresh medium is replaced every 20-28 h. The method for culturing pluripotent stem cells according to claim 1, wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells. The method for culturing pluripotent stem cells as described in any one of claims 1 to 4, which includes the following steps: (S1) using a 48-well plate as a culture container, and the cell seeding density is 4.5~6.5×10 ⁵ cells/mL, The culture volume is 0.4-0.6 mL, and the shaker speed is 170-190 rpm for culture to obtain a cell suspension; (S2) Take the cell suspension obtained in step (S1) and prepare it as a single-cell suspension; ( S3) Take the single cell suspension prepared in step (S2), and repeat steps (S1) and (S2) at least once for culturing. The method for cultivating pluripotent stem cells as described in Claim 5, wherein step (S2) includes: taking the cell mass suspension cultured in step (S1), and sequentially performing enrichment of cell mass, digestion, filtration, centrifugation, and resuspension and optional cell counting, ie preparing the single cell suspension. The method for culturing pluripotent stem cells as described in Claim 5, wherein, in step (S1), the culturing time is 1 to 5 days. The method for cultivating pluripotent stem cells according to claim 5, wherein, in step (S3), the number of repetitions is an integer selected from 1-15. Application of a method for culturing pluripotent stem cells as described in any one of claims 1 to 8 in any one of A1), A2), and A3): A1) expanding the pluripotent stem cells; A2) inducing differentiation of the pluripotent stem cells; A3) Screening an inducer for inducing differentiation of the pluripotent stem cells.

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