CN105950544B - Domestication method of full suspension culture type Marc-145 cell line - Google Patents
Domestication method of full suspension culture type Marc-145 cell line Download PDFInfo
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Abstract
The invention provides a domestication method of a full suspension culture type Marc-145 cell line, which comprises the following steps of (1) culture of adherent culture type Marc-145 cells, (2) domestication of a low serum adherent culture type Marc-145 cell line, (3) domestication of low serum full suspension culture type Marc-145 cells, (4) domestication of serum-free full suspension culture type Marc-145 cells, wherein the cell domestication method can be used for obtaining the Marc-145 cells of suspension culture, the cells are suitable for low serum and serum-free full suspension culture, and the culture density can reach 8 × 10 in a 7.5L reactor according to serum-free culture density6At least 4.4 × 10 can be obtained per ml10The cell number of (3) is equivalent to the cell number harvested by 80-90 15L roller bottles or 18-20 40-layer cell factories, and the invention effect is obvious.
Description
Technical Field
The invention relates to a domestication method of a full suspension culture type Marc-145 cell line.
Background
Marc-145 cells are epithelial cells cloned from rhesus monkey embryonic kidney cells MA-104 by Kim HS in 1993, can grow in an adherent manner after continuous passage, are the most ideal cell strains for researching and producing Porcine reproductive and respiratory syndrome virus (PRRSV, commonly known as blue ear disease) and vaccines at present, and can be used for in vitro proliferation of viruses such as chicken Infectious Bronchitis Virus (IBV) and Porcine Parvovirus (PPV). Because Marc-145 grows in an adherent type, the cells are cultured in an adherent monolayer during virus culture and vaccine production, the amount of the cells is limited by the surface area of a container (such as a cell factory and a roller bottle) or a medium (such as a microcarrier), a large-scale production needs a large enough factory building or expensive microcarriers and a large amount of manual operation, and the bioreactor microcarrier culture still has a plurality of technical problems which are not solved during the amplification culture at present, so the popularization and the application are still difficult. When Marc-145 cells are cultured in an industrial monolayer at the present stage, cells of bovine serum (including fetal bovine serum or newborn bovine serum) with a certain proportion are added into a culture medium to grow normally, the bovine serum is expensive and is derived from natural organisms, the possibility of contaminating exogenous microorganisms and pathogenic factors exists in the process of carrying, collecting and processing the bovine serum, and the use of the bovine serum can cause biological safety risks. In addition, bovine serum is a natural mixture, specific components of the bovine serum are not completely analyzed, the bovine serum causes difficulty in downstream purification processes after being used in the production of biological products, and if residues exist, the bovine serum often causes side reactions of inoculators to influence the product quality. If a serum-free full-suspension process can be adopted for cell culture in the production of biological products, the culture scale is easy to linearly amplify, the process defects caused by cell adherent culture and bovine serum use are avoided, the virus yield and the product quality can be improved, but domestication of a serum-free full-suspension culture type cell strain is one of the most key technical bottlenecks at present. The invention provides a low-serum and serum-free high-density full-suspension culture type Marc-145 cell, a preparation method and an application method thereof, and provides a cell matrix for producing PRRSV vaccine by using the full-suspension culture Marc-145 cell, and the preparation method and the culture method thereof.
Disclosure of Invention
The invention provides a domestication method of a full suspension culture type Marc-145 cell line and provides an expanded culture method of the domesticated full suspension culture type Marc-145 cell.
The invention provides a domestication method of a full suspension culture type Marc-145 cell line, which comprises the following steps:
(1) culture of adherent culture type Marc-145 cells
Selecting an adherent culture type Marc-145 cell which is negative after being detected by bacteria-free mycoplasma and exogenous virus and sensitive to PRRSV, and after the cell grows to be a compact monolayer, using DMEM culture solution containing 10% newborn calf serum to mix according to the ratio of 1: 4 proportion bottle-divided passage, put 5% CO at 37 DEG C2The culture box is used for culturing, and the cells form a compact monolayer within 48-72h, namely the edgeThe cells are flat epithelium cells with clear edges and can be used for domestication under the condition that the cells grow normally;
(2) domestication of low serum adherent culture type Marc-145 cell line
a) And (2) respectively culturing the normally-grown adherent culture type Marc-145 cells in the step (1) for three generations by using DMEM culture solution containing 8% and 5% of newborn bovine serum. The standard for each passage is divided into bottles according to the proportion of 1: 4, and 5% CO is carried out at 37 DEG C2Culturing, and forming a compact monolayer by the cells within 48-72 h;
b) changing the culture solution into DMEM/F12, adding 5% newborn calf serum, and continuing to culture for three generations, wherein the passage standard is the same as the step a);
c) the newborn calf serum in the culture solution is reduced to 3 percent, and the passage ratio is reduced to 1: 3 according to the method in the step b and then the new generation calf serum is cultured for three generations;
d) reducing the content of newborn calf serum in the culture solution to 1%, adding the culture solution with the volume percentage content of 20% and cultured the cell for 48-72h for the last generation into the culture solution, and continuously culturing for five generations, wherein the passage standard is the same as the step c); forming a compact monolayer in 48-72h by the cells, and domesticating to obtain a low serum adherent culture type Marc-145 cell line;
(3) domestication of low-serum full-suspension culture type Marc-145 cells
a) Mixing the low serum adherent culture type Marc-145 cells domesticated in the step (2) with DMEM/F12 and serum-free medium No. 1 according to the volume ratio of 1: 1, adding 3-5% newborn bovine serum to prepare a culture solution, and adjusting the cell density to 3-5 × 10 by using the culture solution5/ml，100-130r/min、37℃ 5％CO2Performing shake culture;
b) the cell density does not reach 1 × 10 by sampling and counting every 48h of culture6When the cell density reaches 1 × 10, the cell density is changed to be 1 through re-suspension culture by using fresh culture solution after 800-6When the cell density reaches 2 × 10, the cell density is cultured in a flask by centrifugation at 1000r/min of 800-6In the case of ml, 800-step centrifugation at 1000r/min is performed according to the proportion of 1: 3, the cell density reaches 2 × 10 after the continuous three-generation culture for 48h according to the proportion of 1: 36When the cell is/ml, domesticating to obtain a low serum full suspension culture type Marc-145 cell line;
(4) domestication of serum-free full-suspension culture type Marc-145 cells
a) Adding 3% newborn bovine serum into serum-free medium No. 2 (Bailing biotechnology, Inc., Lanzhou, product number: BFLM101.05), and directly diluting the low-serum full-suspension culture type Marc-145 cells obtained in step (3) to density of 5-7 × 105The mixture is placed at 100 ℃ and 130r/min and 37 ℃ in 5 percent CO2Performing shake culture;
b) the cell density reaches 3 × 10 by sampling and counting every 24h of culture6For each ml, the cell suspension was directly diluted to 5 × 105The concentration of the newborn calf serum of each generation of culture is reduced by 1 percent, the serum is reduced to 0 by 4 th generation, the culture is completely carried out by serum-free culture, and the cell density reaches 3 × 10 after the continuous third generation culture for 48 hours6And (4) acclimating to obtain a serum-free full-suspension culture type Marc-145 cell line at the point of/ml.
The invention also provides a Marc-145 cell in full suspension culture obtained by the method of claim 1.
The invention also provides a culture method of the full suspension culture type Marc-145 cells in a serum-free culture medium, which comprises the following steps:
a) culturing seed cells, recovering a full suspension culture type Marc-145 cell, adding 5-10% newborn calf serum into 40-60ml serum-free culture medium No. 2, placing at 110r/min and 37 deg.C with 5% CO2Culturing;
when the cells grew to 3 × 106When the concentration is more than ml, the cells are diluted to the density of 5-7 × 10 by using a culture solution of a serum-free culture medium No. 2 plus 1-3% newborn calf serum5The concentration is 250-350ml, 100r/min and 5% CO at 37 DEG C2Culturing;
expanding culture until cell density reaches 3 × 106When the concentration is more than ml, directly diluting the cells with serum-free culture medium No. 2 to the density of 5-7 × 105Continuously expanding culture for one ml, wherein the total volume is 400-600 ml; the culture conditions were: 100-130r/min, 5% CO at 37 DEG C2；
The cell density reaches 3 × 106When the volume is more than ml, transferring the culture medium into a bioreactor for culture;
b) bioreactor batch culture, inoculating No. 2 serum-free culture medium with density of 5-7 × 105Marc-145 cells to bioreactor culture volume, culture conditions were: the rotation speed is 70-120r/min, the temperature is 37 ℃, the dissolved oxygen is 40-70 percent, and the pH value is 7.15-7.25.
The invention also provides a culture method of the full suspension culture type Marc-145 cells in a serum-free culture medium, which comprises the following steps:
inoculating the full suspension culture type Marc-145 cells to a bioreactor for culture by using a serum-free culture medium No. 2, wherein the initial cell density is 5-7 × 105The initial culture volume is 10-20% more than the minimum allowable bioreactor culture volume, and the culture conditions are as follows: the rotating speed is 50-70r/min, the temperature is 37 ℃, the dissolved oxygen is 25-40 percent, and the pH value is 7.15-7.25;
the cell density reaches 2 × 106When the volume is more than ml, serum-free medium No. 2 with the volume equal to the initial culture volume is added, and the culture conditions are changed as follows: the rotating speed is 70-90r/min, the temperature is 37 ℃, the dissolved oxygen is 35-45 percent, and the pH value is 7.15-7.25;
the cell density reaches 4 × 106When the volume is more than ml, the serum-free culture medium No. 2 is supplemented to the highest culture volume, and the culture conditions are changed as follows: the rotation speed is 90-120r/min, the temperature is 37 ℃, the dissolved oxygen is 50-70 percent, and the pH value is 7.10-7.20.
The invention also provides a culture method of the full suspension culture type Marc-145 cells in a low serum culture medium, which comprises the following steps:
inoculating full suspension culture type Marc-145 cells to a bioreactor for culture by using a serum-free culture medium No. 2 and adding 1-3% newborn calf serum, wherein the initial cell density is 5-7 × 105The initial culture volume is 10-20% more than the minimum allowable bioreactor culture volume, and the culture conditions are as follows: the rotating speed is 50-70r/min, the temperature is 37 ℃, the dissolved oxygen is 25-40 percent, and the pH value is 7.15-7.25;
the cell density reaches 2 × 106When the volume is more than ml, serum-free medium No. 2 with the volume equal to the initial culture volume is added, and the culture conditions are changed as follows: the rotating speed is 70-90r/min, the temperature is 37 ℃, the dissolved oxygen is 35-45 percent, and the pH value is 7.15-7.25;
the cell density reaches 6 × 106When the volume is more than ml, the serum-free culture medium No. 2 is supplemented to the highest culture volume, and the culture conditions are changed as follows: the rotation speed is 90-120r/min, the temperature is 37 ℃, the dissolved oxygen is 50-70 percent, and the pH value is 7.10-7.20.
The invention has the beneficial effects that: book (I)The cell acclimation method and the obtained Marc-145 cell capable of being cultured in suspension are suitable for low-serum and serum-free full suspension culture, and the serum-free culture density of the cell can reach 8 × 10 in a 7.5L reactor (the actual maximum culture volume is 5.5L)6At least 4.4 × 10 can be obtained per ml10The number of cells in the bioreactor is equivalent to 80-90 roller bottles with 15L or 18-20 roller bottles with 40 layers, and according to the calculated number, the capacity of a bioreactor with 500L of culture volume is equivalent to the capacity of 300 roller bottles with 30 bottle positions at present. The cells cultured in suspension can be greatly saved in field and manpower input when being applied to actual production, accords with the current concepts of energy conservation, environmental protection and low carbon, brings remarkable economic benefit and also can generate better social benefit.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 shows an adherent culture type Marc-145 cell before acclimation;
FIG. 2 shows domesticated Marc-145 cells in suspension culture;
FIG. 3 is a diagram showing the result of detection of exogenous viruses by an acclimated suspension culture type Marc-145 cell fluorescent antibody method;
FIG. 4 is a graph showing the growth of a suspension culture type Marc-145 cell in a bioreactor without serum culture;
FIG. 5 is a graph showing the growth of the domesticated Marc-145 suspension cultured cells in a bioreactor in low serum culture.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples are commercially available unless otherwise specified.
Example 1
1. Materials:
1) cell lines: marc-145 rhesus monkey embryonic kidney cells, provided by research center of animal cell engineering technology in Gansu province;
2) DMEM medium, Gibco, cat #: 02-5062 EJ;
3) DMEM/F12 medium, Gibco, cat #: 12500-096;
4) serum-free medium No. 1, university lark biotechnology limited, cat #: BFLM 401.05;
5) serum-free medium No. 2, department of biotechnology limited, langzhou, cat #: BFLM 101.05;
6) low serum medium, bazoon biotechnology limited, langzhou, cat #: BLLM 101.05;
7) newborn bovine serum, bio-engineering ltd, national sea of langzhou, lot number: 20150716, respectively;
8) DMSO (dimethyl sulfoxide), Thermo Fisher Scientific Corporation, cat #: 206888);
9) trypsin, Gibco, cat # s: 27250, a 0.25% trypsin solution (EDTA-Na 0.3g/L) was prepared.
2. The instruments and vessels
1) Bioreactor, b.braun, model: BIOSTATEOR B plus, maximum culture volume 5L;
2) shaker, KUHNER, model: ISF 1-XC;
3)CO2incubator, Thermo Fisher Scientific Corporation, model: 3111;
4) centrifuge, Hunan Kaida, model: TD5Z, TDL 80-2B;
5) phase contrast inverted microscope, OLYMPUS CKX 41;
6) fluorescence inverted microscope, OLYMPUS AX71, OLYMPUS;
7) cell culture flasks (Corning, T25 cat No.: 430639, T75 cat No.: 430641);
8) conical cell bottle 150ml, jiangsu haimen, specification: 150 ml;
9) suspension culture flasks 500ml, baoling plastic products ltd, lang, specification: 500 ml;
10) pipettor, BRAND GMBH + CO KG, model: accu-jetORpro.
2. The method comprises the following steps:
1) culture of adherent culture type Marc-145 cells
Selecting one of the adherent culture type Marc-145 cells which are negative in bacteria, mycoplasma and exogenous viruses and sensitive to PRRSV from a cell bank of the research center of animal cell engineering technology in Gansu province for recovery culture, wherein the number of the recovered cells is as follows: GsACC2B0000086, DMEM with 10% newborn calf serum (the following serum concentrations are volume percentage content), culture conditions: 5% CO at 37 ℃2The incubator of (2) for cultivation. After the cells are recovered, the activity is 94.6%, a compact monolayer is formed after the cells are cultured for 48h, the cells are divided into bottles according to the proportion of 1: 4 and subcultured for 48h to form the compact monolayer, the edges of the cells are clear, the shapes are flat, and the cells are epithelial, and the figure 1 shows that the cells are obtained. Cells growing normally can be used for acclimatization.
2) Domestication of low serum adherent culture type Marc-145 cell line
a) Culturing the normally grown adherent culture type Marc-145 cells of 1) for the third generation by using a DMEM culture solution of 8% newborn bovine serum, and dividing bottles according to the ratio of 1: 4 for passage, wherein the culture conditions are as follows: 5% CO at 37 ℃2Culturing, wherein each generation forms a compact monolayer within 48-72 h;
b) b, after the newborn calf serum in the culture solution is reduced to 5%, culturing for three generations according to the method in the step a;
c) c, changing the culture solution into DMEM/F12 and adding 5% newborn calf serum, and continuing to culture the third generation according to the method in the step b;
d) the newborn calf serum in the culture solution is reduced to 3 percent, and the passage proportion is reduced to 1: 3 according to the method in the step c and then the new generation calf serum is cultured for three generations;
e) the newborn calf serum in the culture solution is reduced to 1 percent, the culture solution with the volume percentage content of 20 percent and which is used for culturing the cell for 48 to 72 hours in the previous generation is added into the culture solution (the cell is proved to secrete factors beneficial to the growth of the cell when being cultured with low serum) for continuous culture for five generations, and the culture method is the same as that of d;
f) the cells form a compact monolayer in 48-72h, which shows that the domesticated low serum adherent culture type Marc-145 cell line is adapted, and a part of cells at the stage can be frozen in liquid nitrogen, so that the cells are directly recovered for use when accidents occur in the later domestication process, and the time is saved. According to the conventional method for freezing and storing cells,the formula of the frozen stock solution comprises 80 percent of DMEM/F12, 10 percent of newborn bovine serum and 10 percent of DMSO, and the cell density is 3 × 106The cell viability is 99 percent, and the filling amount of each frozen tube is 1.5 ml.
3) Domestication and establishment of low-serum full-suspension culture type Marc-145 cell line
a) Mixing the domesticated Marc-145 cells cultured in 2) with DMEM/F12 and serum-free medium No. 1 (from Bailing biotechnology, Inc., Lanzhou, Cathao: BFLM401.05) at a volume ratio of 1: 1, adding 3-5% newborn calf serum to obtain culture solution, and adjusting cell density to 4.6 × 10 with the culture solution5Perml, using conical cell flask at 110r/min, 37 deg.C 5% CO2Performing shake culture;
b) the cell density does not reach 1 × 10 by sampling and counting every 48h of culture6The suspension culture is replaced by fresh culture solution after centrifugation at 1000r/min and 800-6When per ml, the cell is centrifuged according to the method and the ratio of 1: 2 and is divided into bottles for culture until the cell density reaches 2 × 106When the cells are cultured for three generations according to the ratio of 1: 3, the cell density reaches 2 × 106At/ml, the low serum full suspension culture type Marc-145 cell line is acclimatized, and a part of the cells at the stage can be frozen by liquid nitrogen. Low serum full suspension acclimation required 10-20 generations.
The culture results and the conditions of the subculture procedures are shown in the table, and the cells of the 16 th generation were cryopreserved by using a cryopreservation solution formula of 80% serum-free medium No. 1 + 10% newborn bovine serum + 10% DMSO, and the cell density was 1.6 × 107The cell activity is 98.1 percent per ml, and the filling amount of each cryopreservation tube is 1.5 ml.
TABLE 1 Marc-145 cell low serum whole suspension culture acclimatization process counting result and passage operation condition table
4) Domestication and establishment of serum-free full-suspension culture type Marc-145 cell line
Marc-145 cells, which were fully suspension-cultured in the 16 th passage of low serum in the above step 3), were directly diluted (without centrifugation) with 3% newborn bovine serum-containing serum-free medium No. 2, and were cultured in a conical cell flask at 5 × 105Inoculating at a density of 110r/min and 5% CO at 37 deg.C2And (5) shaking culture.
The cell density reaches 3 × 10 by sampling and counting every 24h6For each ml, the cell suspension was directly diluted to 5 × 105The serum concentration of newborn calf serum of each generation of culture is reduced by 1 percent, the serum is reduced to 0 when the 4 th generation is reached, the serum-free culture is completely used, and the cell density reaches 3 × 10 when the cell density reaches 48h after the continuous three generations of culture6At/ml, indicating that the serum-free full-suspension culture type Marc-145 cell line is acclimatized, and freezing and storing to establish a main cell bank and a working cell bank. The culture results and the culture medium conditions are shown in Table 2 below, and the cell morphology is shown in FIG. 2.
And the 7 th generation cells are frozen to establish a main cell bank, and the formula of a freezing medium is 80 percent of serum-free culture medium No. 1, 10 percent of newborn bovine serum and 10 percent of DMSO, and the cell density is 1.49 × 107The cell viability is 98.9 percent, the filling amount of each freezing storage tube is 1.5ml, a working cell bank is established by culturing two generations after recovering from the main cell bank, the formula of the freezing storage liquid of the working cell bank is the same as that of the main cell bank, and the cell density is 1.63 × 107The cell viability is 99.3 percent, and the filling amount of each cryopreservation tube is 1.5 ml.
TABLE 2 Marc-145 cell serum-free whole suspension culture acclimatization process counting result and passage operation condition table
5) Assay for full suspension culture type Marc-145 cells
The detection was carried out according to "cell standards for production" of the three 2010 editions of pharmacopoeia of the people's republic of China.
a) And (4) sterile inspection: inoculating the supernatant of the full suspension culture type Marc-145 cell culture into a thioglycollate medium, a tyrose peptone agar medium (inclined plane) and a glucose peptone medium, and culturing all negative cells;
b) and (3) mycoplasma test: the supernatant of the Marc-145 cells in full suspension culture is frozen and thawed once and is inoculated into a mycoplasma liquid culture medium and a mycoplasma solid culture medium to be cultured to be negative;
c) and (3) exogenous virus inspection: inoculating the supernatant of the full suspension culture type Marc-145 cells subjected to freeze thawing once to Vero cells and ST cells for culture, wherein the cells have no pathological changes; the fluorescent antibody method detects Bovine Viral Diarrhea Virus (BVDV) and Porcine Parvovirus (PPV) as negative, as shown in FIG. 3; the red blood cell adsorption test was also negative.
d) Chromosome examination counted 50 chromosomes in metaphase, with 2 n-60 accounting for 84%.
6) Bioreactor high-density culture of serum-free full-suspension culture type Marc-145 cells
a) Seed cell culture recovering a full suspension culture type Marc-145 cell from a working cell bank, adding 5% newborn calf serum into No. 250 ml serum-free culture medium, placing at 110r/min and 37 ℃ with 5% CO2And (5) culturing.
The cell density reaches 3.75 × 10 after 48h of culture6Diluting to 300ml with serum-free medium No. 2 plus 1% newborn calf serum, and performing amplification culture in suspension culture flask to obtain diluted cell density of 6.25 × 105Per ml, culture conditions: 100r/min, 37 ℃ 5% CO2And (5) culturing.
The cell density reaches 4.36 × 10 after 48h of culture6Taking 140ml of cell suspension, adding serum-free medium No. 2 to 1000ml, and performing two suspension culture and expansion culture in 500ml per bottle under the culture conditions: 110r/min, 5% CO at 37 DEG C2Culturing at a cell density of 6.1 × 105/ml。
The cell density reaches 3.91 × 10 after 48h of culture6One part of the mixture is transferred into a bioreactor for culture, and the rest part is 5-7 × 105Continuous generation culture in per ml densityAnd (5) nourishing.
b) Bioreactor batch culture bioreactor preparation following bioreactor protocol, the above Marc-145 cells were grown in full suspension culture in serum-free Medium No. 2 at a density of 6 × 105The culture medium was inoculated in a volume of 5L/ml into a bioreactor. Reactor parameters: the rotating speed is 100r/min, the temperature is 37 ℃, the dissolved oxygen content is 45 percent, and the pH value is 7.22. Cell density was counted every 12 hours. The cell count results are shown in Table 3 and the growth curves are shown in FIG. 4.
c) Bioreactor feed culture bioreactor preparation of bioreactor protocol, the above Marc-145 cells were grown in full suspension at a density of 7 × 10 using serum-free Medium No. 25The culture medium is inoculated into a bioreactor for culture in a volume of 2L, the reactor parameters are that the rotating speed is 60r/min, the temperature is 37 ℃, the dissolved oxygen is 25 percent, the pH value is 7.25, the cell density and the activity are counted every 12, and the cell density reaches 26.3 × 10 after 36h5Adding serum-free culture medium to 4L/ml, changing reactor parameters into rotation speed of 90r/min, temperature of 37 deg.C, dissolved oxygen of 40%, pH7.2, culturing for 72h until cell density reaches 54.3 × 105Adding serum-free culture medium to 5.5L/ml, culturing at a rotation speed of 120r/min, a temperature of 37 deg.C, dissolved oxygen of 60%, pH of 7.10, and culturing for 96 hr until the density reaches 94.2 × 105And/ml. The cell count results are shown in Table 3 and the growth curves are shown in FIG. 4.
In the case of batch culture, the seeding density was 6 × 10 in both culture methods55L Co-inoculation 3 × 10/ml9The cell density of the cells is 8.54 × 10 after 96h of culture6The total volume of the solution/ml reaches 4.27 × 1010About 14 times of growth, 93.1% cell viability, and a fed-batch culture inoculation density of 7 × 105Perml, 2L initial culture volume Co-inoculation 1.4 × 109The cell density of the cells (2) is 9.43 × 10 after 96h of culture6Per ml, the total amount reaches 5.19 × 1010About 37 times of growth, and the cell viability is 98.1%. Therefore, the fed-batch culture is more suitable for bioreactor culture of the full suspension culture type Marc-145 cells.
TABLE 3 serum-free culture counting result table of full suspension culture type Marc-145 cell bioreactor
Note: in the table, the data before "/" are values before culture medium supplementation, and the data after "/" are values after culture medium supplementation.
7) Full suspension culture type Marc-145 cells are cultured in bioreactor with low serum and high density
The culture method is fed-batch culture, and low serum culture medium and 3% newborn calf serum are cultured in a bioreactor according to the method of 6c, and 6.4 × 105The cell density reaches 24.2 × 10 after 36h of inoculation culture at the density of/ml5The serum-free medium is supplemented to 4L/ml, and the cell density reaches 60.4 × 10 after 72h5The serum-free medium is supplemented to 5.5L/ml, and the cell density reaches 105.6 × 10 after 96h5And/ml. The cell count results are shown in Table 4 and the growth curves are shown in FIG. 5.
TABLE 4 Low serum culture counting result table for full suspension culture type Marc-145 cell bioreactor
Note: in the table, the data before "/" are values before culture medium supplementation, and the data after "/" are values after culture medium supplementation.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (1)
1. The domestication method of the full suspension culture type Marc-145 cell line is characterized by comprising the following steps: the method comprises the following steps:
(1) culture of adherent culture type Marc-145 cells
Selecting the materials which are sterilized,Detecting mycoplasma and exogenous viruses as negative, and using an adherent culture type Marc-145 cell sensitive to PRRSV to grow a compact monolayer, and then using a DMEM culture solution containing 10% newborn calf serum to perform cell culture according to the ratio of 1: 4 proportion bottle-divided passage, put 5% CO at 37 DEG C2The culture box is used for culturing, the cells form a compact monolayer within 48-72h, and the compact monolayer is the clear edge squamous epithelial cells which can be used for domestication under the condition that the cells grow normally;
(2) domestication of low serum adherent culture type Marc-145 cell line
a) Culturing the normally-grown adherent culture type Marc-145 cells in the step (1) for three generations by using a DMEM culture solution containing 8% of newborn bovine serum; and (3) reducing the newborn bovine serum in the culture solution to 5%, and culturing for three generations, wherein the standard of each passage is that according to the ratio of 1: 4 proportion of 5% CO at 37 deg.C2Culturing, and forming a compact monolayer by the cells within 48-72 h;
b) changing the culture solution into DMEM/F12, adding 5% newborn calf serum, and continuing to culture for three generations, wherein the passage standard is the same as the step a);
c) and c, reducing the newborn calf serum in the culture solution to 3%, and reducing the passage proportion to 1: 3 culturing for the third generation;
d) reducing the content of newborn calf serum in the culture solution to 1%, adding the culture solution with the volume percentage content of 20% and cultured the cell for 48-72h for the last generation into the culture solution, and continuously culturing for five generations, wherein the passage standard is the same as the step c); forming a compact monolayer in 48-72h by the cells, and domesticating to obtain a low serum adherent culture type Marc-145 cell line;
(3) domestication of low-serum full-suspension culture type Marc-145 cells
a) Mixing the low serum adherent culture type Marc-145 cells domesticated in the step (2) with DMEM/F12 and serum-free medium No. 1 according to the volume ratio of 1: 1, adding 3-5% newborn bovine serum to prepare a culture solution, and adjusting the cell density to 3-5 × 10 by using the culture solution5/ml，100-130r/min、37℃ 5% CO2Performing shake culture;
b) the cell density does not reach 1 × 10 by sampling and counting every 48h of culture6When the cell density reaches 1 × 10, the cell density is changed to be 1 through re-suspension culture by using fresh culture solution after 800-6At/ml, 800-: 2 ratio ofCulturing in bottles until the cell density reaches 2 × 106At the time of/ml, 800-class 1000r/min centrifugation is performed according to the proportion of 1: 3, the cell density reaches 2 × 10 after the continuous three-generation culture for 48h according to the proportion of 1: 36When the cell is/ml, domesticating to obtain a low serum full suspension culture type Marc-145 cell line;
(4) domestication of serum-free full-suspension culture type Marc-145 cells
a) Adding 3% newborn calf serum into serum-free medium No. 2, and directly diluting the low-serum full-suspension culture type Marc-145 cells obtained in step (3) to the density of 5-7 × 105The mixture is placed at 100 ℃ and 130r/min and 37 ℃ in 5 percent CO2Performing shake culture;
b) the cell density reaches 3 × 10 by sampling and counting every 24h of culture6For each ml, the cell suspension was directly diluted to 5 × 105The concentration of the newborn calf serum of each generation of the inoculation culture is reduced by 1 percent, the serum is reduced to 0 by 4 th generation, the new-born calf serum is completely cultured by a serum-free culture medium, and the cell density reaches 3 × 10 after the continuous third generation culture for 48 hours6When the cell is/ml, domesticating to obtain a serum-free full-suspension culture type Marc-145 cell line;
the serum-free medium No. 1 is purchased from Lanzhou Bailing biotechnology limited, and has a product number of: BFLM 401.05;
the serum-free medium No. 2 is purchased from Lanzhou Bailing biotechnology limited, and has a product number of: BFLM 101.05.
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| CN110241089A (en) * | 2019-05-07 | 2019-09-17 | 江苏南农高科技股份有限公司 | A kind of method of full suspension cell culture production porcine reproductive and respiratory syndrome virus antigen |
| CN110373379A (en) * | 2019-07-25 | 2019-10-25 | 北京鼎持生物技术有限公司 | A kind of method that the full suspension cell of Marc-145 directly cultivates PRRS virus vaccine |
| CN110669722A (en) * | 2019-10-23 | 2020-01-10 | 长春生物制品研究所有限责任公司 | Serum-free full-suspension domestication method for MDCK cells |
| CN116694575B (en) * | 2023-08-02 | 2023-12-01 | 上海澳斯康生物制药有限公司 | Method for suspension culture of Marc145 cells |
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