CN116555171A - Domestication method, stem cell line and application of pig muscle stem cells suitable for carrier-free serum-free suspension culture - Google Patents
Domestication method, stem cell line and application of pig muscle stem cells suitable for carrier-free serum-free suspension culture Download PDFInfo
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Abstract
The invention discloses a domestication method, a stem cell line and application of pig muscle stem cells suitable for carrier-free serum-free suspension culture, and belongs to the technical field of cell culture. The domestication method comprises the steps of sequentially carrying out 2D adherent growth, 3D microcarrier adherent suspension culture, 3D microcarrier adherent suspension amplification culture and carrier-free serum-free suspension culture on pig muscle stem cells to obtain a pig muscle stem cell line suitable for carrier-free serum-free suspension culture, domesticating pig immortalized muscle stem cells YP-S4-SC by the method to obtain a young pig muscle stem cell strain YP-S4-S-SC suitable for carrier-free serum-free suspension culture, wherein the stem cell line can be subjected to carrier-free serum-free suspension culture in a shake flask, a transfer flask and a reactor, and the maximum cell density is 1.5x10 6 The cell viability is above 90% and the product can be used for large-scale cell culture meatAnd (3) preparation.
Description
Technical Field
The invention belongs to the technical field of cell culture, and particularly relates to a domestication method, a stem cell line and application of pig muscle stem cells suitable for carrier-free and serum-free suspension culture.
Background
Cell culture meat is to culture animal muscle stem cells in vitro by using cell culture technology, form muscle tissues by differentiation, and process the muscle tissues into edible meat proteins, and belongs to an emerging future food. The cell in-vitro culture mainly comprises two main modes of adherence culture and suspension culture, and compared with the adherence culture, the suspension culture cell has obvious advantages: the suspension cells can grow and proliferate in a suspension state in a culture medium, the cell density can reach tens of millions of cells per milliliter, and the cells are convenient to passage and easy to harvest. However, many cells can only be cultivated by adherence due to the nature of anchored growth, and the number of cells harvested at once is severely limited. For commercial culture modes, it is of primary importance to achieve maximization of cell number. Therefore, it is important to acclimate adherent cell lines to suspension-grown cell lines.
The traditional adherence culture uses animal serum to provide necessary nutrition components and biological factors for cell adherence, proliferation and differentiation, but the serum components are ambiguous, mycoplasma, viruses and the like can be possibly brought into the extracted serum, and the requirements of food safety can not be met. Recent studies show that the growth rate, cell density, product and protein expression level of serum-free medium on cells are comparable to those of serum medium, and proliferation and differentiation nodes of cells can be regulated by precisely controlling the components of serum-free medium, so that the significant advantages of the serum-free medium are gradually replaced by serum-containing cell culture.
Due to the high technical barriers of serum-free suspension growth, few cell lines can be easily transformed from an adherent growth mode to a carrier-free serum-free suspension growth mode. At present, embryonic stem cells have successfully realized suspension culture, and more than 90% of cells can be grown in a single-cell serum-free suspension manner, and mesenchymal stem cells can be grown in a suspension manner in which cells are anchored and clustered. The muscle stem cells are suspended and adhered on microcarriers for culture, and the harvested cells are limited, depend on serum growth and do not meet the commercial requirements of cell culture meat, and no carrier-free and serum-free suspension growth is really realized.
Therefore, there is an urgent need to acclimate muscle stem cells to stem cell lines suitable for carrier-free serum-free suspension growth, and to expand suspension culture to expand a sufficient number of muscle stem cells, which is an important means to achieve commercial production of cell culture meat.
Disclosure of Invention
1. Object of the invention
The invention provides a domestication method, a stem cell line and application of pig muscle stem cells suitable for carrier-free and serum-free suspension culture aiming at the blank of research in the field of the current carrier-free and serum-free suspension culture of muscle stem cells, wherein the domestication method sequentially carries out 2D adherent growth, 3D microcarrier adherent suspension culture, 3D microcarrier adherent suspension amplification culture and carrier-free and serum-free suspension culture on the pig muscle stem cells to obtain the pig muscle stem cell line suitable for carrier-free and serum-free suspension culture, and the pig muscle stem cell YP-S4-SC is domesticated by the method to obtain the young pig muscle stem cell line YP-S4-SC suitable for carrier-free and serum-free suspension culture, and the cell line can carry out carrier-free and serum-free suspension culture in shake flasks, rotating flasks and reactors, and the highest cell density is up to 1.5x10 6 And the cell viability is above 90% and can be used for preparing large-scale cell culture meat.
2. Technical proposal
In order to solve the problems, the technical scheme adopted by the invention is as follows:
the invention provides a domestication method of pig muscle stem cells suitable for carrier-free serum-free suspension culture, the domesticated pig muscle stem cell line can be suitable for carrier-free serum-free suspension culture, the method comprises the following steps:
s1:2D adherent growth of pig muscle stem cells, culturing the pig muscle stem cells in a DMEM culture medium containing 15-20% of fetal calf serum in a 37 ℃ incubator for 2-3 days, culturing by changing liquid, performing pancreatin digestion treatment at the cell density of 60-80%, and centrifuging to obtain cell sediment;
s2:3D microcarrier adherence suspension culture, suspending the cell sediment in S1 by using a DMEM culture medium containing 15% -20% of fetal calf serum, inoculating the cell sediment into a DMEM culture medium rotating bottle containing 1-5 mg/ml of micro-slide glass and 15% -20% of fetal calf serum, and carrying out 3D microcarrier adherence suspension culture; changing 50% of fresh culture medium after 2-4 days; after 5-6 days, adding micro slide cracking liquid (3D) with the final concentration of 1-2 mg/mlDigest solution), cracking for 20-30 min at 50+ -5 rpm in a 37 ℃ incubator, and centrifuging after the micro slide is completely dissolved to obtain cell sediment;
s3:3D microcarrier adherence suspension amplification culture, re-suspending the cell sediment in S2 by using a complete culture medium containing 15% -20% of fetal calf serum, inoculating the cell sediment into a bioreactor containing 1-5 mg/ml of a microcarrier and 15% -20% of fetal calf serum, and carrying out 3D microcarrier adherence suspension amplification culture; changing 50% of fresh culture medium after 2-4 days; after 6-7 days, 50% of the culture medium is removed and a micro-slide lysate (3D) with a final concentration of 1-2 mg/ml is addedDigest solution), and after the microcarrier is completely dissolved, the cells adapting to the adherent suspension are harvested by centrifugation;
s4: and (3) carrying out carrier-free and serum-free suspension culture acclimation, inoculating the cells which are obtained in the step (S3) and are suitable for adherent suspension into a serum-free culture medium containing 0.01% of hydroxypropyl methylcellulose and 0.1% of an anti-caking agent, carrying out suspension acclimation culture, and obtaining the pig muscle stem cell line which is suitable for serum-free suspension culture after stable proliferation.
Preferably, in the step S1, the cells are incubated in DMEM medium containing 15% fetal bovine serum at 37℃for 2 days, and then subjected to pancreatin digestion at 60% cell density.
Preferably, in the step S2, the cell sediment is obtained by centrifugation in the DMEM culture medium heavy suspension S1 containing 15% of fetal calf serum, and the cell sediment is inoculated into a DMEM culture medium rotary bottle containing 1mg/ml of micro-slide glass and 15% of fetal calf serum for 3D microcarrier adherence suspension culture; after 3 days, 50% of fresh medium was changed; after 5 days, 1mg/ml final concentration of the micro slide lysate (3D)Digest solution), lysing for 20-30 min at 50rpm in a 37 ℃ incubator, and centrifuging after complete dissolution of the micro slide to obtain a cell pellet.
Preferably, in the step S2, the cell density after the flask inoculation is (2 to 10). Times.10 4 And each ml.
Preferably, in the step S2, the 3D microcarrier-attached suspension culture conditions are as follows: the magnetic stirring system is controlled at the rotating speed of 40rpm for 5min in a 37 ℃ incubator; 0rpm,2h; after 24 hours, the rotation speed is adjusted to 50rpm, so that cells can be better attached to the microcarrier.
Preferably, in the step S3, the cell sediment in the S2 is resuspended by using a complete medium containing 15% of fetal calf serum, and inoculated into a bioreactor containing 1mg/ml of micro-slide glass and 15% of fetal calf serum for 3D microcarrier adherence suspension amplification culture; after 3 days, 50% of fresh medium was changed; after 6 days, 50% of the medium was removed and 1mg/ml final concentration of the micro slide lysate (3D)Digest solution), after complete dissolution of the microcarriers, the cells adapted for adherent suspension were harvested by centrifugation.
Preferably, in the step S4, the cell density after inoculation in the serum-free medium is (2 to 5). Times.10 5 And each ml.
Preferably, the suspension acclimation culture conditions in the step S4 are as follows: the rotation speed is 90-120 rpm at 37 ℃.
Preferably, the pig muscle stem cells are pig muscle stem cells YP-S4-SC, are preserved in China Center for Type Culture Collection (CCTCC), have a preservation address of university of Wuhan, china, a preservation number of CCTCC NO: C2022256, and a preservation day of 2022, 8 months and 6 days, and can be spontaneously immortalized.
Preferably, the method for domesticating the pig muscle stem cells suitable for carrier-free and serum-free suspension culture comprises the following steps:
s1:2D adherent growth, culturing pig muscle stem cells YP-S4-SC in DMEM medium containing 15% fetal calf serum at 37deg.C for 2 days, changing liquid, performing pancreatin digestion treatment at 60% cell density for 3 days, and centrifuging to obtain cell precipitate;
s2:3D microcarrier adherence suspension culture, re-suspending with DMEM culture medium containing 15% fetal calf serum, inoculating to a rotary bottle containing 1mg/ml microcarrier, and performing 3D microcarrier adherence suspension culture; the cell density after bottle inoculation is (2-10) x 10 4 The conditions of the 3D microcarrier adherence suspension culture are: the magnetic stirring system is controlled at the rotating speed of 40rpm for 5min in a 37 ℃ incubator; 0rpm,2h; the rotating speed is adjusted to 50rpm after 24 hours; after 3 days, 50% of fresh medium was changed; adding microcarrier lysate after 5 days, cracking for 20-30 min at 50rpm in a 37 ℃ incubator, and centrifuging after the microcarrier is completely dissolved to obtain cell sediment;
s3:3D microcarrier adherent suspension amplification culture, re-suspending the cell sediment in S2 by using a complete culture medium containing 15% fetal bovine serum, then inoculating the cell sediment into a bioreactor containing 1mg/ml of a micro-slide and 15% fetal bovine serum, and carrying out 3D microcarrier adherent suspension amplification culture, and replacing 50% of fresh culture medium after 3 days; adding microcarrier lysate after 6 days, cracking for 20-30 min at 50rpm in a 37 ℃ incubator, and centrifuging after the microcarrier is completely dissolved to obtain cell sediment;
s4: and (3) carrying out carrier-free and serum-free suspension culture domestication, inoculating the cells which are harvested in the step (S3) and are suitable for adherent suspension into a serum-free culture medium shake flask containing 0.01% of hydroxypropyl methylcellulose and 0.1% of anti-caking agent, carrying out suspension domestication culture at 37 ℃ and the rotating speed of 90-120 rpm, and obtaining the pig immortalized muscle stem cell line which is suitable for serum-free suspension culture after stable proliferation.
The invention also provides a pig muscle stem cell line which is obtained by the domestication method for the pig muscle stem cell which is suitable for carrier-free and serum-free suspension culture.
The invention also provides a pig muscle stem cell line suitable for carrier-free and serum-free suspension culture, which is characterized in that the pig muscle stem cell line is named as YP-S4-S-SC, and is preserved in China Center for Type Culture Collection (CCTCC), the preservation address is China university of Wuhan, the preservation number is CCTCC NO: C2022372, and the preservation date is 2022, 12 months and 7 days.
The invention also provides a serum-free suspension amplification culture method of the pig muscle stem cell line suitable for carrier-free and serum-free suspension culture, which comprises the following steps of:
serum-free amplification culture is carried out in shake flasks, which comprise 250ml shake flasks and 500ml shake flasks, the rotational speed of the shake flasks is 90-120 rpm, and the initial cell density is (5-25). Times.10 4 Individual/ml;
or carrying out serum-free amplification culture in a rotary bottle comprising 500ml and 1L rotary bottle, wherein the rotary bottle rotation speed is 90-120 rpm, and the initial cell density is (5-25) multiplied by 10 4 And each ml.
The invention also provides application of the pig immortalized muscle stem cell line suitable for carrier-free and serum-free suspension culture.
Preferably, the above-mentioned applications include use in the preparation of cell culture meats.
3. Advantageous effects
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a domestication method, a stem cell line and application of pig muscle stem cells suitable for carrier-free serum-free suspension culture, wherein the domestication method sequentially carries out 2D adherence growth and 3D micro-growth on the pig muscle stem cellsCarrying out carrier adherence suspension culture, 3D micro-carrier adherence suspension amplification culture and carrier-free serum-free suspension culture to obtain a pig muscle stem cell line suitable for carrier-free serum-free suspension culture, and domesticating pig immortalized muscle stem cells YP-S4-SC by the method to obtain a stem cell line YP-S4-S-SC, wherein the cell line can be subjected to carrier-free serum-free suspension culture in a shake flask, a rotary flask and a reactor, and the highest cell density is 1.5x10 6 And the cell viability is above 90% and can be used for preparing large-scale cell culture meat. The suspension proliferation of the pig muscle stem cells breaks the adherent growth characteristic of the muscle stem cells, can save the culture space, the culture cost, the labor cost and the like, and plays an important role in promoting the development of the cultured meat. The stem cell line YP-S4-S-SC provided by the invention is the first pig muscle stem cell line capable of being cultured in a suspending way internationally.
Drawings
FIG. 1 is a photograph of the growth of muscle stem cells over 2D 10 cm.
Figure 2 is a photograph of the growth of muscle stem cells on a 3D microcarrier.
FIG. 3 shows the results of doubling and cell viability of muscle stem cells in serum-free shake flasks for different growth cycles.
Figure 4 is an adherence of muscle stem cells to a plant protein scaffold after serum-free suspension amplification.
FIG. 5 shows the survival of cells on a plant protein scaffold after serum-free suspension amplification of muscle stem cells.
Detailed Description
The invention is further described below in connection with specific embodiments.
The terms such as "upper", "lower", "left", "right", "middle" and the like are also used in the present specification for convenience of description, and are not intended to limit the scope of the present invention, but rather to change or adjust the relative relationship thereof, and are also considered to be within the scope of the present invention without substantial change of technical content.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs; the term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
As used herein, the term "about" is used to provide the flexibility and inaccuracy associated with a given term, metric or value. The degree of flexibility of a particular variable can be readily determined by one skilled in the art.
As used herein, the term "is intended to be synonymous with" one or more of ". For example, "at least one of A, B and C" expressly includes a only, B only, C only, and respective combinations thereof.
Concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a numerical range of about 1 to about 4.5 should be interpreted to include not only the explicitly recited limits of 1 to about 4.5, but also include individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges reciting only one numerical value, such as "less than about 4.5," which should be construed to include all such values and ranges. Moreover, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.
Example 1
The embodiment provides a domestication method of pig muscle stem cells suitable for carrier-free and serum-free suspension culture, which takes spontaneous immortalized cell line pig muscle stem cells YP-S4-SC (preserved in China center for type culture Collection, with preservation address of university of Wuhan, china, with preservation number of CCTCCNO: C2022256, and preservation date of 2022, 8, 6 days) as an original cell line, and comprises the following steps:
s1: the pig muscle stem cells grow in a 2D adherence way, primary pig muscle stem cells YP-S4-SC are taken out of a liquid nitrogen tank, are quickly fused in a water bath kettle at 37 ℃, are added into 4ml of DMEM culture medium (Gibco, C11330500 BT) of 15% fetal bovine serum (Sigma, F8318), are centrifuged for 5min at 330g, and the frozen stock solution is removed to obtain cell sediment; re-suspending cells with DMEM medium containing 15% fetal bovine serum, inoculating the cells into a 10cm culture dish paved with collagen, culturing in a 37 ℃ incubator for 2 days, changing liquid, and performing digestion and passage when the cell density is about 60% for 3 days; as shown in fig. 1, digestion with pancreatin for 2min, then adding serum culture medium to stop digestion, centrifuging for 5min at 330g, and separating to obtain cell precipitate;
s2:3D microcarrier of the pig muscle stem cells are subjected to adherent suspension culture, cell precipitation is resuspended by 10ml of DMEM medium containing 15% fetal calf serum, and 10 μl of the resuspended cell is taken for cell counting; 10ml of 15% foetal calf serum DMEM medium, 60mg of micro slide (Hua Kan, F01-100) are added to a sterile 125ml roller bottle, and are gently shaken to be fully dissolved; placing the cell suspension in 125ml rotary bottle, adding culture medium to 60ml, and cell density of 4×10 4 Placing the mixture in a 37 ℃ incubator, and controlling the rotation speed of a magnetic stirring system to 40rpm for 5min; continuously running at 0rpm for 2 hours to enable the cells to be better attached to the microcarriers, and adjusting the rotating speed to be 50rpm after 24 hours; taking out the rotary bottle on the 3 rd day, stopping stirring for about 5-10 min, fully precipitating the microcarrier, sucking 50% of supernatant from a side port by using a pipetting gun, replacing 50% of fresh culture medium, continuously culturing and observing the proliferation condition of the muscle stem cells on the microcarrier (shown in figure 2); taking out the rotary bottle on day 5, allowing microcarrier to settle for 5-10 min without stirring, sucking out about 2/3 supernatant, and adding microcarrier lysate (3D) with final concentration of 1mg/ml into the rotary bottleDigest solution), the rotary bottle is cracked for 20 to 30 minutes at 50rpm in a 37 ℃ incubator, after the micro slide glass is completely dissolved, 330g is centrifugated for 5 minutes, cell sediment is obtained and resuspended for counting, and the cell proliferation is 10 to 16 times;
s3:3D microcarrier is adhered to the wall for suspension amplification culture, 2g microcarrier slices and 500ml complete culture medium containing 15% fetal bovine serum are added into a sterile bottle, so that the microcarrier slices are completely dissolved; 7.5X10 collected by roller bottle culture 7 Cells were resuspended in 50ml of complete medium containing 15% fetal bovine serum, transferred to sterile flasks and medium was added to a final volume of 2L; sterile vials were connected to bioreactor (Hua Kan, 3D FloTrix with luer connector @ vivaSPIN), all media in the vessel was pumped into the bioreactor at 300ml/min with peristaltic pump, DO set point was set at 70%, pH 7.2, temperature 37 ℃, agitation set at 35rpm, and controller would automatically adjust air and CO 2 Inputs and thermal pads to maintain the system at a set point; on day 3, 1L of waste medium in the container is pumped into a waste bottle at a speed of 300ml/min by a peristaltic pump, and 1L of fresh complete medium containing 15% fetal bovine serum is injected at a speed of 300ml/min by the peristaltic pump; on day 6, pumping 1L of waste medium in the container into a waste bottle at a speed of 300ml/min by a peristaltic pump, and injecting concentrated 3D microcarrier lysate at a speed of 300ml/min by the peristaltic pumpThe final concentration of the digist solution is 1 mg/ml), in the microcarrier dissolution process, the stirring speed is increased to 50rpm, after all microcarriers are completely dissolved, the cell suspension in the culture flask is pumped into a collection flask at the speed of 300ml/min, 330g is centrifuged for 5min, the supernatant is discarded, the suspension is resuspended in 500ml PBS, the sample is used for counting cells, the proliferation of the cells is 10-16 times, the centrifugation is carried out again after the counting is completed, the supernatant is discarded, and the frozen stock is added for cell freezing;
s4: carrier-free serum-free suspension culture acclimation of pig muscle stem cells, resuscitating the cells obtained in S3, placing the cells in 125ml shake flask, adding 20ml serum-free culture medium containing 0.01% hydroxypropyl methylcellulose (Selleck, S4444) and 0.1% anti-caking agent (Gibco, 01-0057 AE), and collecting 1×10 cells 7 Cell density of 5X 10 5 Carrying out suspension domestication at 90rpm and 120rpm per ml; on days 2 and 4, 10ml of serum-free medium was supplemented, and the cells were grown simultaneouslyDetecting conditions and metabolic indexes; on day 6, the cell suspension was collected in a 50ml centrifuge tube, 330g was centrifuged for 5min, the supernatant was recovered, and 4ml of pancreatin substitute TrypLE was added TM Express (Gibco, 12604-021), digested at 37℃for 2min. After adding 6ml of DMEM/F12, the digestion was stopped and the cells were blown off, centrifuged again, 10ml of culture medium was added for resuspension, and the cell suspension was taken for cell counting, and cell proliferation was remarkable. Taking 5×10 according to the counting result 6 Freezing and storing; placing the remaining resuspended cells into shake flasks with a cell density of 5X 10 5 Continuously culturing per ml to obtain a pig muscle stem cell line suitable for carrier-free and serum-free suspension culture, namely a young pig muscle stem cell strain YP-S4-S-SC, which is preserved in China Center for Type Culture Collection (CCTCC), wherein the preservation address is China university of Wuhan, the preservation number is CCTCC NO: C2022372, and the preservation date is 2022, 12 months and 7 days.
Example 2
The present embodiment provides a method for serum-free suspension amplification culture of a pig muscle stem cell line in shake flasks, the culture medium of which can be referred to the Chinese patent entitled "invention" with publication number CN112708591B, specifically comprising the steps of:
(1) Transferring the successfully suspended muscle stem cells of example 1 into shake flask for small scale amplification culture, placing the cells into 125ml/250ml shake flask, adding serum-free medium containing 0.01% hydroxypropyl methylcellulose and 0.1% anti-caking agent, and collecting 1×10 cells 7 Cell density of 2.5X10 5 The rotation speed is 90-120 rpm per ml.
(2) 10ml of serum-free medium was supplemented on days 2 and 4, and the growth and metabolic index of the cells were examined.
(3) On day 6, the cell suspension was collected in a 50ml centrifuge tube, centrifuged at 330g for 5min, the supernatant was recovered, 4ml of pancreatin substitute (Gibco, 12604-021) was added, and digested at 37℃for 2min. Adding 6ml DMEM/F12, stopping digestion, blowing off cells, centrifuging again, adding 10ml culture solution for resuspension, taking cell suspension for cell counting, and the cell activity rate is above 90%.
(4) Calculating total cells based on the counting resultCounting, calculating cell multiplication time, performing digestion and passage every 6 days, with cell activity rate above 90%, and cell density of 2.5X10 after shake flask inoculation 5 The maximum density of cells in the whole process of the expansion culture reaches (1-2) multiplied by 10 6 Cells proliferated stably for at least 8 passages per ml (as shown in FIG. 3).
Example 3
The present embodiment provides serum-free suspension amplification culture of porcine muscle stem cell lines in roller bottles suitable for carrier-free serum-free suspension culture, and the culture medium can refer to Chinese patent publication No. CN112708591B, which specifically comprises the following steps:
(1) The muscle stem cells successfully suspended in example 1 were transferred to a flask for small scale up culture, the cells were placed in a 500ml flask, 200ml of serum-free medium containing 0.01% hydroxypropyl methylcellulose and 0.1% anti-clumping agent was added, and the cells were 1.6X10 7 Cell density of 8X 10 4 The rotation speed is 90-120 rpm per ml.
(2) 100ml of serum-free medium was supplemented on days 2 and 4, and the growth and metabolic index of the cells were examined.
(3) On day 6, the cell suspension was collected in a 50ml centrifuge tube, centrifuged at 330g for 5min, the supernatant was recovered, 4ml of pancreatin substitute was added, and digested at 37℃for 2min. After adding 6ml of DMEM/F12, the cells were digested and blown off, centrifuged again, 10ml of culture medium was added for resuspension, and the cell suspension was taken for cell counting with a cell viability of 99% or higher (Table 1).
TABLE 1 suspension amplification of YP-S4-S-SC in roller bottle culture results
Example 4
The present embodiment provides carrier-free serum-free suspension amplification culture of porcine muscle stem cell lines in a bioreactor, the culture medium can refer to Chinese issued patent with publication number CN112708591B, and specifically comprises the following steps:
(1) Transferring the successfully suspended muscle stem cells into a 5L bioreactor for amplification culture, and culturing 8×10 7 The cells were added 1.2L (0.5 mM calcium ion) of serum-free medium containing 0.01% hydroxypropyl methylcellulose and 0.1% anti-clumping agent, and the cell density was 6.7X10 4 The rotation speed is 90-120 rpm per ml.
(2) 600ml of serum-free medium was supplemented on days 2 and 4, and the growth and metabolic index of the cells were examined.
(3) On day 6, the cell suspension was collected in a 50ml centrifuge tube, centrifuged at 330g for 5min, the supernatant was recovered, and pancreatin substitute was added and digested at 37℃for 2min. DMEM/F12 was added to terminate digestion and blow off the cells, centrifugation was performed again, 10ml of culture medium was added to resuspend, and cell suspension was taken for cell counting with cell viability above 98% (Table 2).
TABLE 2 suspension-amplified culture results of YP-S4-S-SC in bioreactor
Example 5
The embodiment provides application of a pig muscle stem cell line suitable for carrier-free serum-free suspension culture in preparation of cell culture meat, and the application is mainly used for evaluating the re-adherence capacity of the pig muscle stem cell line on a plant peanut protein bracket, and specifically comprises the following steps:
(1) Preparing a peanut protein scaffold: soaking peanut protein scaffold in ultrapure water at room temperature until it is completely expanded, cutting into pieces with size of 2cm×2cm×0.5cm with a scalpel, vacuum drying at 60deg.C for 3h, sterilizing at 121deg.C under high temperature and high pressure for 15min, soaking sterilized scaffold in serum-free culture medium, and placing in a 37 deg.C incubator for use.
(2) Cell inoculation: clamping the peanut protein bracket soaked in the serum-free culture medium into a low-adhesion culture dish by using forceps, and slightly squeezing to remove excessive culture solution; 200, 300, 400. Mu.L of cell suspension was taken and the cell density was 1.25X10 7 Uniformly inoculating cells/mu L on a peanut protein bracket; after cell inoculation is completed, the cells are transferred into a 37 ℃ incubator to stand for 2 hours, and the cells are migrated to the branchAfter the incubation, 30mL of serum-free medium was added to the dish and the incubation was continued in an incubator at 37 ℃.
(3) Drop condition of cells on peanut protein scaffold: serum-free medium soaked in scaffolds was aspirated daily, centrifuged at 300g, and cell counts were performed and daily cell drop was recorded (Table 3).
TABLE 3 shedding of cells of different densities onto plant protein scaffolds
(4) Cell adhesion ability and cell survival were examined under a microscope: the adherence of the muscle stem cells on the peanut protein scaffold is observed and recorded under a microscope (shown in fig. 4), and the survival of the muscle stem cells on the peanut protein scaffold is observed and recorded under a microscope (shown in fig. 5), which shows that the young pig muscle stem cell strain YP-S4-S-SC which is suitable for carrier-free serum-free suspension culture can grow on the surface of the protein scaffold to prepare cell culture meat.
Claims (9)
1. The domestication method of the pig muscle stem cells suitable for carrier-free and serum-free suspension culture is characterized in that the pig muscle stem cells obtained by domestication by the domestication method can be subjected to carrier-free and serum-free suspension culture, and the domestication method comprises the following steps:
s1:2D adherent growth of pig muscle stem cells, culturing the pig muscle stem cells in a DMEM culture medium containing 15-20% of fetal calf serum in a 37 ℃ incubator for 2-3 days, culturing by changing liquid, performing pancreatin digestion treatment at the cell density of 60-80%, and centrifuging to obtain cell sediment;
s2:3D microcarrier adherence suspension culture, suspending the cell sediment in S1 by using a DMEM culture medium containing 15% -20% of fetal calf serum, inoculating the cell sediment into a DMEM culture medium rotating bottle containing 1-5 mg/ml of micro-slide glass and 15% -20% of fetal calf serum, and carrying out 3D microcarrier adherence suspension culture; changing 50% of fresh culture medium after 2-4 days; removing 50% of culture medium after 5-6 days, adding a micro-slide lysate with the final concentration of 1-2 mg/ml, cracking for 20-30 min at 50+ -5 rpm in a 37 ℃ incubator, and centrifuging after the micro-slide is completely dissolved to obtain cell sediment;
s3:3D microcarrier adherence suspension amplification culture, re-suspending cell sediment in S2 by using a complete culture medium containing 15% -20% of fetal calf serum, inoculating the cell sediment into a bioreactor containing 1-5 mg/ml of a micro-slide and a DMEM culture medium containing 15% -20% of fetal calf serum, and carrying out 3D microcarrier adherence suspension amplification culture; changing 50% of fresh culture medium after 2-4 days; removing 50% of culture medium after 6-7 days, adding a micro-slide lysate with the final concentration of 1-2 mg/ml, and centrifuging to harvest cells suitable for wall-attached suspension after the micro-carriers are completely dissolved;
s4: and (3) carrying out carrier-free and serum-free suspension culture acclimation, inoculating the cells which are obtained in the step (S3) and are suitable for adherent suspension into a serum-free culture medium containing 0.01% of hydroxypropyl methylcellulose and 0.1% of an anti-caking agent, carrying out suspension acclimation culture, and obtaining the pig muscle stem cell line which is suitable for serum-free suspension culture after stable proliferation.
2. The method for acclimatizing a pig muscle stem cell adapted to a carrier-free serum-free suspension culture according to claim 1, wherein the method comprises the steps of
In the step S1, a DMEM culture medium containing 15% of fetal calf serum is used for culturing for 2 days in a 37 ℃ incubator, liquid exchange is carried out, and pancreatin digestion treatment is carried out at the cell density of 60%;
and/or in the step S2, the cell sediment is obtained by centrifugation in the DMEM culture medium heavy suspension S1 containing 15% of fetal calf serum, and the cell sediment is inoculated into a DMEM culture medium rotary bottle containing 1mg/ml of micro-slide glass and 15% of fetal calf serum for 3D microcarrier adherence suspension culture; after 3 days, 50% of fresh medium was changed; after 5 days, adding a micro-slide lysate with the final concentration of 1mg/ml, carrying out lysis for 20-30 min at 50rpm in a 37 ℃ incubator, and centrifuging after the micro-slide is completely dissolved to obtain a cell precipitate;
and/or in the step S3, the cell sediment in S2 is resuspended by DMEM containing 15% fetal calf serum, and inoculated into a bioreactor containing 1mg/ml micro-slide and a complete culture medium containing 15% fetal calf serum for 3D microcarrier adherence suspension amplification culture; after 3 days, 50% of fresh medium was changed; after 6 days, 50% of the medium was removed and 1mg/ml final concentration of microcarrier lysate was added, and after complete dissolution of microcarriers, cells adapted to adherent suspension were harvested by centrifugation.
3. The method for acclimatizing pig muscle stem cells suitable for carrier-free serum-free suspension culture according to claim 2, wherein in the step S2, the cell density after bottle inoculation is (2-10) x 10 4 Individual/ml; the 3D microcarrier adherence suspension culture conditions are as follows: the magnetic stirring system is controlled at the rotating speed of 40rpm for 5min in a 37 ℃ incubator; 0rpm,2h; after 24 hours the rotation speed was adjusted to 50rpm.
4. The method for acclimatizing porcine muscle stem cells suitable for carrier-free serum-free suspension culture according to claim 2 or 3, wherein in the step S4, the cell density after inoculation in serum-free medium is (2-5). Times.10 5 Individual/ml; the suspension domestication culture conditions in the step S4 are as follows: the rotation speed is 90-120 rpm at 37 ℃.
5. The domestication method of the pig muscle stem cells suitable for carrier-free and serum-free suspension culture according to claim 4, wherein the pig muscle stem cells are pig muscle stem cells YP-S4-SC, and are preserved in China center for type culture Collection, with a preservation address of university of Wuhan, china, a preservation number of CCTCCNO: C2022256, and a preservation date of 2022, 8 months and 6 days.
6. A porcine muscle stem cell line adapted for carrier-free serum-free suspension culture, characterized in that it is obtained by acclimation according to the acclimation method of any one of claims 1-5.
7. The swine muscle stem cell line suitable for carrier-free and serum-free suspension culture of claim 6, wherein the swine muscle stem cell line suitable for carrier-free and serum-free suspension culture is named as young swine muscle stem cell strain YP-S4-S-SC, and is preserved in China center for type culture Collection with the preservation address of university of Wuhan, china, with the preservation number of CCTCCNO: C2022372 and the preservation date of 2022, 12 months and 7 days.
8. The pig muscle stem cell line suitable for carrier-free and serum-free suspension culture is characterized by being named as a young pig muscle stem cell strain YP-S4-S-SC, being preserved in China center for type culture Collection, having a preservation address of university of Wuhan in China, a preservation number of CCTCCNO: C2022372, and a preservation date of 2022, 12 months and 7 days.
9. A method of acclimatizing a porcine muscle stem cell line adapted for carrier-free serum-free suspension culture according to any one of claims 1 to 5 and/or a use of a porcine muscle stem cell line adapted for carrier-free serum-free suspension culture according to any one of claims 6 to 8 for the preparation of cell culture meat.
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