Feeder-free, serum-free bovine embryonic stem cell culture medium, culture system and culture method
By using feeder-free, serum-free bovine embryonic stem cell culture media and gelatin-coated culture dishes, combined with the use of IWR-1 and activin A, the problems of high cost and loss of morphological characteristics in bovine embryonic stem cell culture were solved, achieving efficient and stable cell passage and maintenance of pluripotency.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- NORTHWEST A & F UNIV
- Filing Date
- 2025-10-14
- Publication Date
- 2026-04-10
AI Technical Summary
Existing bovine embryonic stem cell culture methods rely on mouse fetal fibroblasts as a feeder layer, which has problems such as cumbersome preparation, large batch-to-batch variability and limited gene manipulation. Furthermore, existing feeder-free culture methods have made limited progress in bovine embryonic stem cells, resulting in high culture costs and loss of morphological characteristics.
Using a feeder-free, serum-free bovine embryonic stem cell culture medium containing IWR-1 and activin A, combined with gelatin-coated culture dishes, a conditioned medium was prepared and cells were passaged through specific steps to ensure the undifferentiated state of the cells and efficient passage.
It significantly reduced the cost of extracellular matrix, improved the passage efficiency and stability of bovine embryonic stem cells, enhanced cell availability, facilitated subsequent gene editing operations, and maintained the undifferentiated characteristics and pluripotency of cells.
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Figure CN120966740B_ABST
Abstract
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:
[0007] 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.
[0008] The conditioned medium is prepared by culturing mouse fetal fibroblasts in an embryonic stem cell basic medium mTeSR1.
[0009] 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 boundary 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 tight connection 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.
[0010] Further, the conditioned medium is prepared by the following steps:
[0011] 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;
[0012] The MEF complete culture medium is DMEM / F12 containing 10%-15% fetal bovine serum by volume.
[0013] 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.
[0014] Further, the concentration of Y27632 in the subculture medium is 10 μM.
[0015] 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:
[0016] coated with the gelatin with the mass concentration of 0.1%-0.2%, and the coated culture dish is placed in a 37℃ incubator for incubation for 30-60 minutes to obtain a gelatin-coated culture dish;
[0017] The bovine embryonic stem cells are cultured in the gelatin-coated culture dish for 3-5 days, and the culture medium is the bovine embryonic stem cell culture medium;
[0018] After the culture is completed, the old culture medium is aspirated and discarded, DPBS is added for cleaning, and then digestion is performed; after the digestion is completed, twice the volume of the bovine embryonic stem cell culture medium is added to terminate the digestion, centrifugation is performed, and the cell precipitate is collected;
[0019] The cell precipitate is resuspended and mixed evenly with the subculture medium, the cell suspension is evenly distributed into new gelatin-coated culture dishes, and the culture dishes are shaken horizontally and crosswise to make the cells evenly distributed; after 24-26 hours, new bovine embryonic stem cell culture medium is replaced, and then new bovine embryonic stem cell culture medium is replaced every day until cloning is formed.
[0020] Further, the culture condition is 37℃ and 5% CO2.
[0021] Further, the cell suspension is evenly distributed into 3-4 new gelatin-coated culture dishes.
[0022] The present application has the following beneficial effects:
[0023] 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 realizing 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, and these two components 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.
[0024] The technical advantages and innovation of the present application make it have broad application prospects in the field of embryonic stem cell technology. Its high efficiency, economy and easy implementation provide strong technical support for related research and application, and have very high practical value and feasibility. BRIEF DESCRIPTION OF DRAWINGS
[0025] Figure 1Morphology of bovine embryonic stem cells cultured without a feeder layer. A: Passage 2 bovine embryonic stem cells without a feeder layer under 4× field of view; B: Passage 2 bovine embryonic stem cells without a feeder layer under 10× field of view; C: Passage 30 bovine embryonic stem cells without a feeder layer under 4× field of view; D: Passage 30 bovine embryonic stem cells without a feeder layer under 10× field of view; Scale bar: 50 μm.
[0026] Figure 2 Alkaline phosphatase (AP) staining of bovine embryonic stem cells cultured without feeder layer; scale bar: 20 μm.
[0027] Figure 3 Immunofluorescence staining images of OCT4, E-cadherin, and NANOG cells in bovine embryonic stem cells cultured without a feeder layer.
[0028] 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.
[0029] Figure 5 Image of chromosome karyotype analysis of bovine embryonic stem cells cultured without feeder layer.
[0030] 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.
[0031] Figure 7 Image of mRNA gene expression in the three germ layers of the embryoid body.
[0032] Figure 8 Morphology of bovine embryonic stem cells cultured without feeder layer under different culture systems; Scale bar: 50 μm. Detailed Implementation
[0033] 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.
[0034] Currently, the mouse and human ESCs culture system without feeder layer is relatively mature, and the ESCs cultured without feeder layer are conducive to improving the gene editing efficiency and developing clinical cell therapy products. The culture dish under the feeder-free culture system needs to be coated with extracellular matrix for adherent growth. However, the research progress of the feeder-free culture of livestock ESCs is very limited. The stable feeder-free culture of livestock ESCs is conducive to the preparation of stem cell breeding and gene editing animals.
[0035] The fibronectin-coated culture dish can be used for the feeder-free culture of bEPSCs and has pluripotency. The culture dish coated with vitronectin or Matrigel can be used for the culture of bESCs, but the morphology is single-layer 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 blank. Compared with the extracellular matrix such as vitronectin, Matrigel, fibronectin and laminin, gelatin has a lower price.
[0036] At present, a stable culture method of bESCs without feeder layer has not been developed. Therefore, the present application provides a bovine embryonic stem cell culture medium without feeder layer and serum, a culture system and a culture method, so as to optimize the culture conditions of bESCs without feeder layer.
[0037] The materials used in the following examples and sources are as follows:
[0038] mTeSR1: purchased from STEMCELL Technologies, item number 85850.
[0039] IWR-1: purchased from Sigma-Alrdich, item number I0161.
[0040] Activin A: purchased from STEMCELL Technologies, item number 78001.
[0041] Y27632: purchased from TOCRIS bioscience, item number 1254.
[0042] 0.1% gelatin aqueous solution: purchased from STEMCELL Technologies, item number 07903.
[0043] Serum-free cell freezing medium: purchased from Suzhou Xinsaimai Biotechnology Co., Ltd., item number C40100.
[0044] DMEM / F12: purchased from Wuhan Punsai Life Science Co., Ltd., item number PM150312.
[0045] Special fetal bovine serum: purchased from Wuhan Punsai Life Science Co., Ltd., item number 164210.
[0046] PBS buffer (1X): purchased from Wuhan Pons Life Science Co., Ltd., product code PB180327.
[0047] Accutase: purchased from Thermo Fisher Scientific, product code A1110501.
[0048] Example 1: Culture of feeder-free bovine embryonic stem cells
[0049] 1. Preparation of conditioned medium
[0050] Mouse embryonic fibroblasts (MEFs) were recovered into a T175 cell culture flask, and when the cells were in good condition and the density reached 80%, the old culture medium was discarded, washed twice with DPBS, and replaced with embryonic stem cell basal medium mTeSR1. The medium collected after 24 hours was the mouse embryonic fibroblast conditioned medium (MEF-CM), which was collected continuously for 3 times. After the collection of MEF-CM was completed, it was filtered with a 0.22 μM filter, and 20 mL / tube was aliquoted and stored at -20°C. The complete medium for MEFs was DMEM / F12 + 10% fetal bovine serum (FBS, volume concentration).
[0051] 2. Coating of culture dishes
[0052] A 0.1% gelatin aqueous solution was taken according to 1 mL / 35 mm cell culture dish, cross-shaking mixed, and a gelatin-coated 35 mm cell culture dish was obtained. Before use, the coated culture plate was incubated in a 37°C incubator for 30 min, and when used, the coating liquid was discarded and DPBS was gently rinsed to absorb the unbound material.
[0053] 3. Passage of feeder-free bovine embryonic stem cells
[0054] Generally, the cells were passaged at a ratio of 1:3-1:4 after 3-4 days of culture, and the specific passage time and passage ratio were determined 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, they must be passaged. Taking a 35 mm cell culture dish as an example:
[0055] (1) Bovine embryonic stem cells were cultured in a gelatin-coated dish at 37°C, 5% CO2 for 3 days, 8 10 4 cells were inoculated in a 35 mm cell culture dish. The culture medium was feeder-free bovine embryonic stem cell complete medium, which was 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 was 2.5 μM, and the concentration of activin A was 25 ng / mL.
[0056] (2) Gently suck out the old medium in 1), wash twice with 1 mL DPBS slowly; add 1 mL of digestive enzyme Accutase and incubate in a 37°C incubator for 2 min; add twice the volume of Accutase to the complete medium without feeder layer bovine embryonic stem cells, and then transfer the cells to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 min;
[0057] (3) After centrifugation, resuspend the cell pellet in the subculture medium, and evenly distribute the cell suspension into gelatin-coated 35 mm cell culture dishes at a ratio of 1:4; shake horizontally and crosswise for 6 times, and then place in a 37°C, 5% CO2 incubator for culture; replace the medium with new complete medium without feeder layer bovine embryonic stem cells after 24 hours, and then replace the medium every day. The subculture medium is based on the complete medium without feeder layer bovine embryonic stem cells and contains Y27632, and the concentration of Y27632 in the subculture medium is 10 μM.
[0058] 4. Cryopreservation and recovery of feeder-free bovine embryonic stem cells
[0059] (1) Cryopreservation of feeder-free bovine embryonic stem cells
[0060] After cell digestion, collect the cell suspension, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the cell pellet in commercial serum-free cell freezing medium, mix gently, and then transfer to a labeled freezing tube. Place the freezing tube in a freezing box in a -80°C refrigerator for overnight cryopreservation, and then transfer to a liquid nitrogen tank for long-term storage. The freezing medium is a commercial serum-free cell freezing medium.
[0061] (2) Recovery of feeder-free bovine embryonic stem cells
[0062] Preheat the water bath to 37°C, take out one frozen bovine embryonic stem cell (500 μL), and place it in a 37°C water bath. Gently shake by hand, and thaw within 1 min. Observe the cell suspension under a microscope when the ice crystals in the cell suspension have completely disappeared. Wipe the surface of the freezing tube with 75% alcohol dust-free paper, 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 complete medium without feeder layer bovine embryonic stem cells 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 horizontally and crosswise for 6 times, and then place in a 37°C, 5% CO2 incubator for culture. Replace the medium with new complete medium without feeder layer bovine embryonic stem cells after 24 hours, and then replace the medium every day until the clones grow to a certain morphology.
[0063] Example 2: Identification of feeder-free bovine embryonic stem cells (FF-bESCs)
[0064] 1. Morphological observation and alkaline phosphatase staining of FF-bESCs
[0065] According to the culture and identification of Example 1, Step 1-Step 3, 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 high nuclear-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 passage process, as shown in Figure 1 .
[0066] After the FF-bESCs colony is formed, the embryonic stem cell original culture solution to be detected is discarded, washed with DPBS for 2 times, fixed with 4% paraformaldehyde at room temperature for 1-2 min, and the subsequent operation steps are performed according to the alkaline phosphatase color reagent kit (Bi Yun Tian, C3206) instruction manual. Incubate at room temperature for about 10-30 min in the dark, stop the color reaction, and observe under a microscope and take pictures. The results are shown in Figure 2 , and the AP staining results show that the FF-bESCs alkaline phosphatase staining is positive.
[0067] 2. Expression analysis of pluripotent marker genes of FF-bESCs
[0068] After extracting the total RNA of the second and thirtieth generations of feeder-free bovine embryonic stem cells and reverse transcribing the cDNA, the expression of pluripotent genes OCT4, SOX2, and NANOG of FF-bESCs is detected by RT-qPCR. There is no significant difference between the mRNA expression amounts of pluripotent genes OCT4, SOX2, and NANOG between the second and thirtieth generations of feeder-free bovine embryonic stem cells, and there is no significant difference between the mRNA expression amounts of OCT4, SOX2, and NANOG between the thirtieth generation and the feeder bovine embryonic stem cells Figure 4 .
[0069] When the confluence of FF-bESCs reaches about 80%, the cells are fixed with 4% PFA for 10 min, and then immunofluorescence staining is performed on the cells to detect the expression of pluripotent marker proteins OCT4, NANOG, and E-cadherin. The results are shown in Figure 3 , and the OCT4, NANOG, and E-cadherin proteins are normally expressed (see Figure 3 ).
[0070] 3. Chromosome karyotype analysis and in vitro differentiation ability analysis of FF-bESCs
[0071] Chromosomal karyotype analysis was performed on FF-bESCs to determine whether the chromosome number was normal. The results showed that the chromosome number was normal, 2n = 60 (see...). Figure 5 ).
[0072] FF-bESCs were digested into single cells and seeded at 5000 cells / well in U-shaped culture plates with ultra-low adsorption density. The cells were cultured in suspension for 6 days, with medium changes every two days. After 6 days, the embryoids were transferred to gelatin-coated 24-well plates for adherent differentiation for 9–14 days. In vitro differentiation results showed that FF-bESCs could differentiate into three germ layer cell types, expressing the ectoderm marker βIII-tubulin, the mesodermal marker α-SMA, and the endoderm marker AFP. Furthermore, RT-qPCR analysis of the three germ layer marker genes showed that all three germ layer marker genes were significantly upregulated. Figure 6 , Figure 7 ).
[0073] Example 3: Optimization of feeder-free bovine embryonic stem cell (FF-bESCs) culture system
[0074] The FF-bESCs culture medium of this invention uses mTeSR1 as the conditioned medium for collection, supplemented with 2.5 μM IWR-1 and 25 ng / mL activin A. Under this system, embryonic stem cells grow in colonies, with raised colonies, smooth edges, high refractive index, blurred cell boundaries within the colonies, high nucleocytoplasmic ratio, and prominent nucleoli. Removal of IWR-1 from the culture medium resulted in mostly monolayer cell growth, losing typical embryonic stem cell morphological characteristics. Removal of activin A from the culture medium caused the embryonic stem cell colonies to flatten. These results indicate that IWR-1 and activin A are crucial for maintaining the undifferentiated state of FF-bESCs. The above images show similar results obtained in at least three independent experiments (see...). Figure 8 ).
[0075] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.
[0076] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.
Claims
1. A product for feeder-free, serum-free culture of bovine embryonic stem cells, characterized in that, The culture medium for bovine embryonic stem cells is free of feeder layer and serum, and the gelatin with a mass concentration of 0.1%-0.2% is used to provide an attachment matrix for the bovine embryonic stem cells; the subculture medium is the bovine embryonic stem cell culture medium containing Y27632; the concentration of Y27632 in the subculture medium is 10 μM. The bovine embryonic stem cell culture medium comprises a conditioned medium and additives, and the additives are IWR-1 and activin A, and the concentrations of the IWR-1 and activin A in the conditioned medium are 2.5 μM and 25 ng / mL, respectively. The conditioned medium is prepared by culturing mouse fetal fibroblasts in embryonic stem cell basal medium mTeSR1. The conditioned medium is prepared by the following steps: The mouse fetal fibroblasts are cultured in MEF complete medium to a density of 80%, the old medium is discarded, DPBS is used for washing, and the embryonic stem cell basal medium mTeSR1 is replaced, and the medium collected after 24-26 hours is the conditioned medium. The MEF complete medium is DMEM / F12 containing 10%-15% fetal bovine serum by volume.
2. A method for culturing bovine embryonic stem cells without a feeder layer and serum, characterized by, The culture method is performed by using the product of claim 1, and the culture method comprises the following steps: The culture dish is coated with the gelatin with a mass concentration of 0.1%-0.2%, and the coated culture dish is incubated at 37°C for 30-60 min to obtain a gelatin-coated culture dish; The bovine embryonic stem cells are cultured in the gelatin-coated culture dish for 3-5 days, and the culture medium is the bovine embryonic stem cell culture medium; After the culture, the old medium is aspirated and discarded, DPBS is added for washing, and then digestion is performed, the digestion is terminated by adding two volumes of the bovine embryonic stem cell culture medium, centrifugation is performed, and the cell precipitate is collected; The cell precipitate is resuspended and mixed uniformly by using the subculture medium, the cell suspension is evenly distributed to new gelatin-coated culture dishes, the culture dishes are shaken horizontally and crosswise to make the cells uniformly distributed, and the bovine embryonic stem cell culture medium is replaced after 24-26 hours, and then the bovine embryonic stem cell culture medium is replaced every day until the clones are formed.
3. The method of claim 2, wherein the bovine embryonic stem cells are cultured in the absence of a feeder layer and serum. The digestion is performed by using Accutase.
4. The method of claim 3, wherein the bovine embryonic stem cells are cultured in the absence of a feeder layer and serum. The culture condition is 37°C and 5% CO2.
5. The method of claim 4, wherein the bovine embryonic stem cells are cultured in the absence of a feeder layer and serum. The cell suspension is evenly distributed to 3-4 new gelatin-coated culture dishes. The digestion is performed by using Accutase. The culture condition is 37°C and 5% CO2. The cell suspension is evenly distributed to 3-4 new gelatin-coated culture dishes.