Feed-layer-free and serum-free bovine embryonic stem cell culture medium, culture system and culture method

Abstract

The invention belongs to the technical field of cell biology, and particularly relates to a feeder-layer-free and serum-free bovine embryonic stem cell culture medium, a culture system and a culture method. The bovine embryonic stem cell culture medium comprises a conditioned culture medium and additives, the additives are IWR-1 and activin A, and the concentrations of the IWR-1 and the activin A in the conditioned culture medium are 2.5-5 [mu] M and 20-25 ng / mL respectively; the culture system comprises a bovine embryonic stem cell culture medium, gelatin with the mass concentration of 0.1-0.2% and a subculture medium, and the gelatin with the mass concentration of 0.1-0.2% is used for providing an attachment matrix for bovine embryonic stem cells; the subculture medium is prepared by adding Y27632 into a bovine embryonic stem cell culture medium. The cost of the extracellular matrix is reduced, and the feeder-layer-free bovine embryonic stem cell has high single cell passage efficiency and is convenient for gene editing operation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cell biology, and particularly relates to a feeder-free and serum-free bovine embryonic stem cell culture medium, a culture system and a culture method. BACKGROUND

[0002] Embryonic stem cells (ESCs) are a kind of cells isolated from the inner cell mass in the blastocyst stage, which can self-renew, proliferate indefinitely in vitro and have the ability to differentiate into three germ layer cells in vivo and in vitro.

[0003] In the past five years, although the establishment of bovine embryonic stem cell lines has made a breakthrough, its culture has long relied on mouse fetal fibroblasts and other feeder cells to provide an attachment interface and secrete specific factors to maintain the characteristics of stem cells. The feeder culture method has played an important role in embryonic stem cell research, but it has inherent defects such as complicated preparation, large batch differences and limited gene manipulation, which seriously restricts the standardization of stem cell research and clinical translation. Feeder-free culture of ESCs is conducive to reducing the cost of culture and promoting the in-depth study of the pluripotency regulation mechanism of ESCs.

[0004] Feeder-free (FF) culture of embryonic stem cells usually uses extracellular matrix such as vitronectin, matrigel, fibronectin and laminin to coat the culture dish. However, the research progress of feeder-free culture of bovine embryonic stem cells (bESCs) is limited. Fibronectin-coated culture dishes can be used for feeder-free culture of bEPSCs and have pluripotency. Vitronectin or matrigel-coated culture dishes can be used for culture of bESCs, but the morphology is single-layer cell growth, which loses the typical morphological characteristics of embryonic stem cells. SUMMARY

[0005] To solve the problems existing in the prior art, the present application provides a feeder-free and serum-free bovine embryonic stem cell culture medium, a culture system and a culture method.

[0006] The present application specifically adopts the following technical solutions: In a first aspect, the present application provides a feeder-free and serum-free bovine embryonic stem cell culture medium, which comprises a conditioned medium and an additive, the additive being IWR-1 and activin A, and the concentrations of the IWR-1 and the activin A in the conditioned medium being 2.5-5 μM and 20 ng / mL-25 ng / mL, respectively. The conditioned medium is prepared by culturing mouse fetal fibroblasts in an embryonic stem cell basic medium mTeSR1.

[0007] During the experiment of the present application, we carefully adjusted the components of the feeder-free and serum-free bovine embryonic stem cell culture medium to explore its influence on the growth state of bovine embryonic stem cells. The experimental results showed that when IWR-1 was removed from the culture medium, the growth pattern of the cells changed significantly, and most of the cells showed a monolayer growth pattern, which was obviously different from the typical morphological characteristics of embryonic stem cells. The original tight aggregation, clear colony structure gradually disappeared, the cell morphology tended to be flat and dispersed, and the three-dimensional sense and tightness characteristic of embryonic stem cells were lost. Similarly, when activin A was removed from the culture medium, the embryonic stem cell colonies also showed a flattening trend. The originally full and three-dimensional colony structure became flat, and the close contact between cells seemed to be weakened. These experimental results strongly indicate that IWR-1 and activin A play a crucial role in maintaining the undifferentiated state of embryonic stem cells. Their presence is indispensable to maintain the typical morphology and undifferentiated characteristics of embryonic stem cells. Once these two key factors are lacking, embryonic stem cells will quickly lose their hallmark features of undifferentiated state, thereby affecting their subsequent biological functions and application value. Therefore, the present application further confirms the key position of IWR-1 and activin A in embryonic stem cell culture, providing important theoretical basis and practical guidance for in vitro culture of embryonic stem cells and related research.

[0008] Further, the conditioned medium is prepared by the following steps: The mouse fetal fibroblasts are cultured in MEF complete culture medium to a density of 80%, the old culture medium is discarded, DPBS is used for washing, and the embryonic stem cell basic culture medium mTeSR1 is replaced. The culture medium collected after 24-26 hours is the conditioned medium; The MEF complete culture medium is DMEM / F12 containing 10%-15% fetal bovine serum by volume.

[0009] In the second aspect, the present application provides a feeder-free and serum-free bovine embryonic stem cell culture system, which comprises the bovine embryonic stem cell culture medium, 0.1%-0.2% gelatin by mass concentration, and a subculture medium. The 0.1%-0.2% gelatin by mass concentration is used to provide an attachment substrate for bovine embryonic stem cells. The subculture medium is the bovine embryonic stem cell culture medium containing Y27632.

[0010] Further, the concentration of Y27632 in the subculture medium is 10 μM.

[0011] In the third aspect, the present application provides a feeder-free and serum-free bovine embryonic stem cell culture method, which is carried out by using the culture system. The culture method comprises the following steps: Coat the culture dish with the gelatin with a mass concentration of 0.1% to 0.2%, place the coated culture dish in a 37℃ incubator, and incubate for 30min to 60min to obtain a gelatin-coated culture dish; Culture the bovine embryonic stem cells in the gelatin-coated culture dish for 3 to 5 days, and the culture medium is the bovine embryonic stem cell culture medium; After the culture is completed, discard the old culture medium, wash with DPBS, then digest, add twice the volume of the bovine embryonic stem cell culture medium to terminate the digestion after the digestion is completed, centrifuge, and collect the cell precipitate; Resuspend and mix the cell precipitate with the subculture medium, evenly distribute the cell suspension into new gelatin-coated culture dishes, and shake horizontally to make the cells evenly distributed; replace the new bovine embryonic stem cell culture medium after 24 to 26 hours, and replace the new bovine embryonic stem cell culture medium every day until the clones are formed.

[0012] Further, the culture condition is 37℃ and 5% CO2.

[0013] Further, the cell suspension is evenly distributed into 3 to 4 new gelatin-coated culture dishes.

[0014] The present application has the following beneficial effects: The present application has made a major breakthrough in the field of embryonic stem cell technology. Through innovative technical means, the present application significantly reduces the cost of extracellular matrix, while achieving efficient single-cell subculture of feeder-free bovine embryonic stem cells, greatly improving the subculture efficiency of cells. In addition, the present application ingeniously adds IWR-1 and activin A in the culture system, which are key factors for maintaining the undifferentiated state of bovine embryonic stem cells (FF-bESCs). This innovation not only improves the stability and availability of cells, but also provides great convenience for subsequent gene editing operations.

[0015] The technical advantages and innovation of the present application make it have broad application prospects in the field of embryonic stem cell technology. Its efficient, economical and easy-to-implement characteristics provide strong technical support for related research and application, and have very high practical value and feasibility. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 : Morphology of feeder-free cultured bovine embryonic stem cells. A is the 2nd generation of feeder-free bovine embryonic stem cells under 4x field, B is the 2nd generation of feeder-free bovine embryonic stem cells under 10x field, C is the 30th generation of feeder-free bovine embryonic stem cells under 4x field, and D is the 30th generation of feeder-free bovine embryonic stem cells under 10x field; scale: 50um.

[0017] Figure 2Alkaline phosphatase (AP) staining of bovine embryonic stem cells cultured without feeder layer; scale bar: 20 μm.

[0018] Figure 3 Immunofluorescence staining images of OCT4, E-cadherin, and NANOG cells in bovine embryonic stem cells cultured without a feeder layer.

[0019] Figure 4 Images showing the expression of pluripotency marker genes OCT4, SOX2, and NANOG in generation 2 and generation 30 bovine embryonic stem cells without feeder layer.

[0020] Figure 5 Image of chromosome karyotype analysis of bovine embryonic stem cells cultured without feeder layer.

[0021] Figure 6 Immunofluorescence staining images of embryoid trilamin cell markers β-III-Tubulin, α-SMA, and AFP; Scale bars: β-III-Tubulin scale bar is 20 μm, SMA and AFP scale bars are 50 μm.

[0022] Figure 7 Image of mRNA gene expression in the three germ layers of the embryoid body.

[0023] Figure 8 Morphology of bovine embryonic stem cells cultured without feeder layer under different culture systems; Scale bar: 50 μm. Detailed Implementation

[0024] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0025] Currently, feeder-free culture systems for mouse and human ESCs are relatively mature. Feeder-free cultured ESCs are beneficial for improving gene editing efficiency and developing clinical-grade cell therapy products. In feeder-free culture systems, culture dishes require additional extracellular matrix coating for adherence and growth. However, research progress on feeder-free culture of livestock ESCs is very limited. Stable feeder-free culture of livestock ESCs is beneficial for stem cell breeding and the preparation of gene-edited animals.

[0026] Fibronectin-coated culture dishes can be used for the culture of bEPSCs without feeder cells and have pluripotency. Vitronectin or Matrigel-coated culture dishes can be used for the culture of bESCs, but their morphology is monolayer cell growth. It is worth noting that although gelatin is widely used for the culture of mouse embryonic stem cells, its application in the feeder-free system of bovine embryonic stem cells is still a blank. Compared with extracellular matrix such as vitronectin, matrigel, fibronectin and laminin, gelatin is cheaper.

[0027] At present, a stable culture method of feeder-free bESCs has not been developed. Therefore, the present application provides a feeder-free and serum-free bovine embryonic stem cell culture medium, culture system and culture method, so as to optimize the culture conditions of bESCs without feeder cells.

[0028] The materials used in the following examples and sources are as follows: mTeSR1: purchased from STEMCELL Technologies, item number 85850.

[0029] IWR-1: purchased from Sigma-Alrdich, item number I0161.

[0030] Activin A: purchased from STEMCELL Technologies, item number 78001.

[0031] Y27632: purchased from TOCRIS bioscience, item number 1254.

[0032] 0.1% gelatin aqueous solution: purchased from STEMCELL Technologies, item number 07903.

[0033] Serum-free cell freezing medium: purchased from Suzhou Xinsaimai Biotechnology Co., Ltd., item number C40100.

[0034] DMEM / F12: purchased from Wuhan Punsai Life Science Co., Ltd., item number PM150312.

[0035] Special fetal bovine serum: purchased from Wuhan Punsai Life Science Co., Ltd., item number 164210.

[0036] PBS buffer (1X): purchased from Wuhan Punsai Life Science Co., Ltd., item number PB180327.

[0037] Accutase: purchased from Thermo Fisher Scientific, item number A1110501.

[0038] Example 1: Culture of feeder-free bovine embryonic stem cells 1. Preparation of conditioned medium Resuscitate mouse embryonic fibroblasts (MEFs) into T175 cell culture flask, when the cell state is good and the density reaches 80%, discard the old culture medium, wash twice with DPBS, replace with embryonic stem cell basal medium mTeSR1, collect the medium after 24 hours, which is mouse embryonic fibroblast conditioned medium (MEF-CM), collect for 3 times in succession. After the collection of MEF-CM is completed, filter with a 0.22 μM filter, 20 mL / tube, and store at -20℃. The complete medium for MEFs is DMEM / F12+10% fetal bovine serum (FBS, volume concentration).

[0039] 2. Coating of culture dish Take the gelatin aqueous solution with a mass concentration of 0.1% according to 1 mL / 35 mm cell culture dish, cross-shake to mix, and obtain a gelatin-coated 35 mm cell culture dish. Incubate the coated culture plate in a 37℃ incubator for 30 min before use, discard the coating liquid, and gently rinse with DPBS to absorb the unbound material.

[0040] 3. Subculture of feeder-free bovine embryonic stem cells Generally, the cells are subcultured according to 1:3-1:4 after 3-4 days of culture, and the specific subculture time and subculture ratio need to be judged according to the cell morphology and density. It should be noted that when the embryonic stem cell clones are large or are about to fuse, subculture must be performed. Take a 35 mm cell culture dish as an example:

[0041] (1) Culture bovine embryonic stem cells in a gelatin-coated culture dish at 37℃, 5% CO2 for 3 days, 8 10 4 cells are inoculated in a 35 mm cell culture dish. The culture medium is feeder-free bovine embryonic stem cell complete medium, which is based on MEF-CM and added with IWR-1 and activin A. The concentration of IWR-1 in the feeder-free bovine embryonic stem cell complete medium is 2.5 μM, and the concentration of activin A is 25 ng / mL;

[0042] (2) Gently aspirate the old culture medium in 1), wash twice with 1 mL DPBS, add 1 mL of digestion enzyme Accutase, and incubate in a 37℃ incubator for 2 min. Add twice the volume of feeder-free bovine embryonic stem cell complete medium to terminate the digestion of the cells, and then transfer to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 min; (3) After centrifugation, resuspend the cell pellet with the subculture medium, and evenly distribute the cell suspension into the gelatin-coated 35 mm cell culture dishes at a ratio of 1:4. Shake the dishes horizontally and crosswise for 6 times, and then place them in a 37℃, 5% CO2 incubator. After 24 hours, replace the medium with fresh feeder-free bovine embryonic stem cell complete medium, and then replace the medium every day.

[0043] 4. Cryopreservation and recovery of feeder-free bovine embryonic stem cells (1) Cryopreservation of feeder-free bovine embryonic stem cells After the cells are digested, collect the cell suspension, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the cell pellet with commercial serum-free cell cryopreservation solution, mix gently, and then transfer it to a labeled cryopreservation tube. Place the cryopreservation tube in a cryopreservation box in a -80℃ refrigerator for overnight cryopreservation, and then transfer it to a liquid nitrogen tank for long-term storage. The cryopreservation solution is commercial serum-free cell cryopreservation solution.

[0044] (2) Recovery of feeder-free bovine embryonic stem cells Preheat the water bath to 37℃, take out one cryopreserved bovine embryonic stem cell sample (500 μL), and place it in the 37℃ water bath. Shake it gently by hand within 1 min, and then take it out when the ice crystals in the cell suspension almost completely disappear. Wipe the surface of the cryopreservation tube with 75% alcohol, and then transfer it to a clean bench. Move the cell suspension to a previously prepared 15 mL centrifuge tube, and then add 4.5 mL of feeder-free bovine embryonic stem cell complete medium drop by drop. Mix the cells gently, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 2 mL of subculture medium, mix the cell suspension, and then transfer it to a new gelatin-coated culture dish. Shake it horizontally and crosswise for 6 times, and then place it in a 37℃, 5% CO2 incubator. After 24 hours, replace the medium with fresh feeder-free bovine embryonic stem cell complete medium, and then replace the medium every day until the clones grow to a certain morphology.

[0045] Example 2: Identification of feeder-free bovine embryonic stem cells (FF-bESCs) 1. Morphological observation and alkaline phosphatase staining of FF-bESCs According to the culture and identification of steps 1-3 in Example 1, it can be seen that FF-bESCs grow in a colony-like manner, the colony is raised, the colony edge is smooth and has high light refraction, the cell boundary in the colony is blurred, the cell has a high nucleus-cytoplasm ratio, and the nucleolus is obvious. The morphological characteristics are similar to those of the initial state of ESCs, and no differentiation phenomenon occurs in the long-term subculture process, as shown in Figure 1 .

[0046] After FF-bESCs colony formation, the embryonic stem cell original culture medium to be tested was discarded, washed twice with DPBS, and fixed with 4% paraformaldehyde at room temperature for 1-2 min. The subsequent operation steps were performed according to the instructions of the alkaline phosphatase color reagent kit (Bi Yun Tian, C3206). Incubate at room temperature for 10-30 min in the dark, stop the color reaction, and then observe under a microscope and take pictures. The results are shown in Figure 2 The AP staining results show that FF-bESCs are positive for alkaline phosphatase staining.

[0047] 2. Analysis of the expression of pluripotent marker genes of FF-bESCs After extracting total RNA from feeder-free bovine embryonic stem cells of passages 2 and 30 and reverse transcribing the cDNA, the expression of pluripotent genes OCT4, SOX2, and NANOG of FF-bESCs was detected by RT-qPCR. There was no significant difference in the mRNA expression of pluripotent genes OCT4, SOX2, and NANOG between feeder-free bovine embryonic stem cells of passages 2 and 30, and there was no significant difference in the mRNA expression of OCT4, SOX2, and NANOG between feeder-free bovine embryonic stem cells of passage 30 and feeder-dependent bovine embryonic stem cells. Figure 4 ).

[0048] When the confluence of FF-bESCs reached about 80%, the cells were fixed with 4% PFA for 10 min, and then immunofluorescence staining was performed to detect the expression of pluripotent marker proteins OCT4, NANOG, and E-cadherin. The results are shown in Figure 3 OCT4, NANOG, and E-cadherin proteins were normally expressed (see Figure 3 ).

[0049] 3. Chromosome karyotype analysis and in vitro differentiation ability analysis of FF-bESCs Chromosome karyotype analysis was performed on FF-bESCs to identify whether the chromosome number was normal. The results showed that the chromosome number of the cells was normal, 2n = 60 (see Figure 5 ).

[0050] FF-bESCs were digested into single cells and seeded in ultra-low attachment density U-shaped culture plates at a density of 5000 cells per well. The cells were cultured in suspension for 6 days, with medium replacement every two days. After 6 days, the embryoid bodies were transferred to gelatin-coated 24-well plates for adherent differentiation culture for 9-14 days. The results of in vitro differentiation showed that FF-bESCs could differentiate into cells of all three germ layers, expressing ectodermal marker βIII-tubulin, mesodermal marker α-SMA, and endodermal marker AFP. In addition, RT-qPCR analysis of the three germ layer marker genes showed that the three germ layer marker genes were significantly upregulated Figure 6 .Figure 7 ).

[0051] Example 3: Optimization of feeder-free bovine embryonic stem cell (FF-bESCs) culture system The FF-bESCs culture medium was based on mTeSR1 medium for conditioned medium collection, and 2.5 μΜ IWR-1 and 25 ng / mL Activin A were added. Under this system, the embryonic stem cells grew in colony form, with raised colonies, smooth edges, high refractive index, fuzzy boundaries between cells, high nuclear-cytoplasmic ratio, and obvious nucleoli. After removing IWR-1 from the medium, most of the cells grew in monolayer, losing the typical embryonic stem cell morphological characteristics. After removing Activin A from the medium, the embryonic stem cell colonies became flat. The above results show that IWR-1 and Activin A are the keys to maintaining the undifferentiated state of FF-bESCs. The above images gave similar results in at least three independent experiments (see Figure 8 ).

[0052] It should be noted that when the present application claims a numerical range, both endpoints of the numerical range and any number between the two endpoints can be selected. In order to prevent repetition, the present application describes preferred embodiments.

[0053] Although preferred embodiments of the present application have been described, those skilled in the art will appreciate that other modifications and alterations to the embodiments are possible. Accordingly, the appended claims are intended to cover all such modifications and alterations as falling within the scope of the present application.

Claims

1. A bovine embryonic stem cell culture medium without a feeder layer and without serum, characterized in that, The medium includes conditioned medium and additives, wherein the additives are IWR-1 and activator A, and the concentrations of IWR-1 and activator A in the conditioned medium are 2.5~5μM and 20ng / mL~25ng / mL, respectively. The conditioned medium was prepared by culturing mouse fetal fibroblasts using the embryonic stem cell basal medium mTeSR1.

2. The bovine embryonic stem cell culture medium without feeder layer and serum according to claim 1, characterized in that, The conditioned medium is prepared using the following steps: Mouse fetal fibroblasts were cultured in MEF complete medium until the density reached 80%. The old medium was discarded, and the cells were washed with DPBS and replaced with embryonic stem cell basal medium mTeSR1. The medium collected after 24-26 hours was the conditioned medium. The complete culture medium for MEFs is DMEM / F12 containing 10%~15% fetal bovine serum by volume.

3. A bovine embryonic stem cell culture system without a feeder layer and without serum, characterized in that, The invention includes the bovine embryonic stem cell culture medium as described in any one of claims 1 to 2, as well as gelatin and passage medium with a mass concentration of 0.1% to 0.2%, wherein the gelatin with a mass concentration of 0.1% to 0.2% is used to provide an attachment matrix for the bovine embryonic stem cells; and the passage medium is the bovine embryonic stem cell culture medium containing Y27632.

4. The bovine embryonic stem cell culture system without feeder layer and serum as described in claim 3, characterized in that, The concentration of Y27632 in the passaged culture medium was 10 μM.

5. A method for culturing bovine embryonic stem cells without a feeder layer or serum, characterized in that, The culture method, using the culture system described in claim 3, comprises the following steps: The culture dish was coated with gelatin at a mass concentration of 0.1%~0.2%, and the coated culture dish was incubated at 37°C for 30min~60min to obtain gelatin-coated culture dish; Bovine embryonic stem cells were cultured in gelatin-coated culture dishes for 3-5 days in the bovine embryonic stem cell culture medium described above. After culturing, the old culture medium was discarded, DPBS was added for washing, and then digestion was performed. After digestion, twice the volume of the bovine embryonic stem cell culture medium was added to stop the digestion. The cells were centrifuged and the cell pellet was collected. Resuspend and mix the cell pellet in the passaged culture medium. Distribute the cell suspension evenly to a new gelatin-coated culture dish and gently shake it horizontally in a cross shape to ensure uniform cell distribution. Replace the culture medium with a new bovine embryonic stem cell culture medium after 24-26 hours, and then replace it with a new bovine embryonic stem cell culture medium daily until clones are formed.

6. The method for culturing bovine embryonic stem cells without a feeder layer and without serum according to claim 5, characterized in that, Accutase was used for digestion.

7. The method for culturing bovine embryonic stem cells without a feeder layer and without serum according to claim 5, characterized in that, The culture conditions were 37℃ and 5% CO2.

8. The method for culturing bovine embryonic stem cells without a feeder layer and without serum according to claim 5, characterized in that, The cell suspension was evenly distributed into 3-4 new gelatin-coated culture dishes.

Images