CN114774369A - Serum-free medium for adenovirus production and adenovirus production method - Google Patents
Serum-free medium for adenovirus production and adenovirus production method Download PDFInfo
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Abstract
The invention relates to a serum-free culture medium for adenovirus production and an adenovirus production method, wherein the culture medium comprises amino acid, vitamin, inorganic salt, glucose, PF68, sodium hyaluronate and water; wherein the vitamins comprise choline chloride, calcium pantothenate, nicotinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, and inositol. The invention adopts pyridoxine hydrochloride to replace folic acid and pyridoxine hydrochloride, forms a vitamin source with choline chloride, calcium pantothenate, nicotinamide, riboflavin, thiamine hydrochloride and inositol, and forms a specific culture medium formula together with PF68 and sodium hyaluronate with amino acid, inorganic salt and glucose, and the accessed adenovirus can efficiently transfect host cells in the process of producing the adenovirus by adopting the culture medium formula.
Description
Technical Field
The invention relates to the technical field of adenovirus vaccines, in particular to a serum-free culture medium for adenovirus production and an adenovirus production method.
Background
The adenovirus is a five-envelope double-strand DNA virus and can infect birds such as chickens, turkeys, ducks, geese and the like, and the types of the adenovirus which is shaped at present mainly comprise: class I, class II and class III. The inactivated avian adenovirus vaccine for preventing adenovirus mainly comprises a tissue inactivated vaccine, a chick embryo inactivated vaccine and a cell inactivated vaccine. Wherein: the tissue inactivated vaccine is prepared by performing chloroform extraction and formaldehyde inactivation on liver homogenate of infected chicken, and the vaccine is simple and extensive in process and cannot be applied to large-scale production; the chicken embryo inactivated vaccine is prepared by inoculating SPF chicken embryo with adenovirus, harvesting allantoic fluid, and inactivating to obtain the final product vaccine, wherein the vaccine has low potency and high cost, and the chicken embryo fibroblast inactivated vaccine has the problems of complex process, high cost and low potency.
Currently available methods for adenovirus production include culturing adenovirus using host cells (e.g., adherent LMH cells), harvesting the virus fluid, inactivating it, and preparing the final vaccine. The production method comprises a host cell proliferation culture step and an adenovirus culture step using the host cell, wherein the latter step is an adenovirus production step.
Traditionally, the culture medium for culturing adenovirus by using host cells is mainly a synthetic medium, such as a common DMEM culture medium, and the like, wherein the synthetic medium contains amino acids, sugars, vitamins, inorganic salts, trace components and the like necessary for cell growth, and in order to avoid the influence of unknown components on cell culture products, the synthetic medium is not added with animal serum, and belongs to a chemically defined culture medium.
However, in the production of adenovirus using conventional synthetic media, the infection efficiency of host cells with adenovirus needs to be improved.
Disclosure of Invention
In view of this, the object of the present invention consists in providing a serum-free medium for the production of adenoviruses, in which the host cells are inoculated with the adenovirus, which has a high infection efficiency with respect to the host cells.
The purpose of the invention can be realized by the following technical scheme:
in a first aspect of the invention, a serum-free medium for adenovirus production is provided, the medium comprising 599.3mg/L-2226mg/L amino acids, 6.5mg/L-49.8mg/L vitamins, 5550.8mg/L-13845.3mg/L inorganic salts, 2150mg/L-5670mg/L glucose, 500mg/L-2000mg/L PF68, 1.2mg/L-7.5mg/L sodium hyaluronate, and water;
wherein the vitamins comprise choline chloride, calcium pantothenate, nicotinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, and inositol.
In some embodiments of the invention, the choline chloride is at a concentration of 0.9mg/L to 7mg/L, the calcium pantothenate is at a concentration of 0.8mg/L to 8.2mg/L, the nicotinamide is at a concentration of 1.3mg/L to 7.8mg/L, the pyridoxine hydrochloride is at a concentration of 0.9mg/L to 8.4mg/L, the riboflavin is at a concentration of 0.2mg/L to 0.7mg/L, the thiamine hydrochloride is at a concentration of 3.7mg/L to 10mg/L, and the inositol is at a concentration of 1.7mg/L to 12 mg/L.
In some embodiments of the invention, the amino acid comprises one or more of glycine, arginine, L-cystine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, L-tyrosine, and valine.
In some embodiments of the invention, the glycine is at a concentration of 21mg/L to 48.3mg/L, the arginine is at a concentration of 28.8mg/L to 105.8mg/L, the L-cystine is at a concentration of 18.1mg/L to 79.4mg/L, the glutamine is at a concentration of 208.8mg/L to 735.8mg/L, the histidine is at a concentration of 29.4mg/L to 52.9mg/L, the isoleucine is at a concentration of 30.5mg/L to 132.3mg/L, the leucine is at a concentration of 33.5mg/L to 144.9mg/L, the lysine is at a concentration of 22.2mg/L to 184mg/L, the methionine is at a concentration of 21mg/L to 37.8mg/L, and the phenylalanine is at a concentration of 26.2mg/L to 83.2mg/L, the concentration of the serine is 29.4mg/L-52.9mg/L, the concentration of the threonine is 46.5mg/L-119.7mg/L, the concentration of the tryptophan is 11.2mg/L-20.2mg/L, the concentration of the L-tyrosine is 32.8mg/L-131mg/L, and the concentration of the valine is 35.8mg/L-118.4 mg/L.
In some embodiments of the invention, the inorganic salt comprises one or more of calcium chloride, iron nitrate nonahydrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, and sodium dihydrogen phosphate monohydrate.
In some embodiments of the invention, the concentration of calcium chloride is 184.8mg/L-332.6mg/L, the concentration of iron nitrate nonahydrate is 0.1mg/L-2.1mg/L, the concentration of magnesium sulfate is 28.4mg/L-123.1mg/L, the concentration of potassium chloride is 180mg/L-504mg/L, the concentration of sodium bicarbonate is 2590mg/L-4662mg/L, the concentration of sodium chloride is 2480mg/L-8064mg/L, and the concentration of sodium dihydrogen phosphate monohydrate is 87.5mg/L-157.5 mg/L.
In some embodiments of the invention, the medium comprises 45mg/L to 48.3mg/L glycine, 100mg/L to 105.8mg/L arginine, 78.1mg/L to 79.4mg/L L-cystine, 600mg/L to 735.8mg/L glutamine, 49mg/L to 52.9mg/L histidine, 130mg/L to 132.3mg/L isoleucine, 140mg/L to 144.9mg/L leucine, 180mg/L to 184mg/L lysine, 35mg/L to 37.8mg/L methionine, 80mg/L to 83.2mg/L phenylalanine, 49mg/L to 52.9mg/L serine, 110mg/L to 119.7mg/L threonine, 19mg/L-20.2mg/L tryptophan, 125mg/L-131 mg/LL-tyrosine, 110mg/L-118.4mg/L valine, 6.5mg/L-7mg/L choline chloride, 6mg/L-7.5mg/L calcium pantothenate, 7.3mg/L-7.8mg/L nicotinamide, 7mg/L-8.4mg/L pyridoxine hydrochloride, 0.65mg/L-0.7mg/L riboflavin, 0.7mg/L-6.7mg/L thiamine hydrochloride, 11.5mg/L-12mg/L inositol, 310mg/L-332.6mg/L calcium chloride, 0.1mg/L-1.1mg/L ferric nitrate nonahydrate, 115mg/L-123.1mg/L magnesium sulfate, 500mg/L-504mg/L potassium chloride, 4300mg/L-4662mg/L sodium bicarbonate 7500mg/L-8064mg/L sodium chloride, 150mg/L-157.5mg/L monobasic sodium phosphate, 5200mg/L-5670mg/L glucose, 1900mg/L-2000mg/L PF68, 7mg/L-7.5mg/L sodium hyaluronate and water.
In a second aspect of the present invention, there is provided a method for producing an adenovirus, the method comprising the steps of: providing a host cell, placing the host cell in the culture medium of the first aspect, inoculating adenovirus, culturing, and harvesting the adenovirus.
In some embodiments of the invention, the culturing is for a period of 72h to 96 h.
In some embodiments of the invention, the host cell is placed in the culture medium and cultured for 72h to 96h prior to inoculation with the adenovirus.
In some embodiments of the invention, the density of the host cells in the culture medium is 2 × 106cells/mL-6×106cells/mL。
In some embodiments of the invention, the adenovirus is administered at a concentration of 1MOI to 10 MOI.
In some embodiments of the invention, the host cell is HEK 293.
Compared with the prior art, the invention has the following beneficial effects:
aiming at the defects of the traditional adenovirus culture medium (especially a DMEM culture medium), the vitamin source does not contain folic acid, pyridoxine hydrochloride, choline chloride, calcium pantothenate, nicotinamide, riboflavin, thiamine hydrochloride and inositol are adopted to form the vitamin source, PF68 and sodium hyaluronate are added in a matching way without adding sodium pyruvate, and a specific culture medium formula is formed together with amino acid, inorganic salt and glucose, and the inoculated adenovirus can efficiently transfect host cells (such as HEK293) in the process of producing the adenovirus by adopting the culture medium formula.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and the advantages thereof, the drawings that are required in the description of the embodiments will be briefly introduced below. It is obvious that the drawings in the following description are only some embodiments of the application, and that for a person skilled in the art, other drawings can also be derived from them without inventive effort.
FIG. 1 shows the result of 18h fluorescence detection after inoculation of DMEM medium in accordance with example 5 of the present invention;
FIG. 2 shows the results of 18h fluorescence detection after inoculation of the medium of formula 1 in example 5;
FIG. 3 shows the results of 18h fluorescence detection after inoculation of the medium of formula 2 in example 5 of the present invention;
FIG. 4 shows the results of 18h fluorescence detection after inoculation of the medium of formula 3 in example 5 of the present invention;
FIG. 5 shows the 18h fluorescence detection result of the medium inoculation in the formula 4 in example 5 of the present invention;
FIG. 6 shows the 18h fluorescence detection result of the medium inoculation in the control formula 1 in example 5 of the present invention;
FIG. 7 shows the 18h fluorescence detection result of the medium inoculation in the control formula 2 in example 5 of the present invention;
FIG. 8 shows the 18h fluorescence detection result of the medium inoculation in the control formula 3 in example 5 of the present invention;
FIG. 9 shows the 18h fluorescence detection result of the culture medium inoculated with the virus in the control formula 4 in example 5 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the drawings, embodiments and examples. It should be understood that these embodiments and examples are given solely for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention, which is provided for the purpose of providing a more thorough understanding of the present disclosure. It should also be understood that the present invention can be embodied in many different forms and is not limited to the embodiments and examples described herein, and that various changes or modifications may be effected therein by one of ordinary skill in the art without departing from the spirit and scope of the invention and the resulting equivalents are intended to be within the scope of the present application. Furthermore, in the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present invention, and it is to be understood that the present invention may be practiced without one or more of these details.
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 terminology used herein in the description of the invention is for the purpose of describing particular embodiments and examples only and is not intended to be limiting of the invention.
Term(s)
Unless otherwise indicated or contradicted, terms or phrases used herein have the following meanings:
the term "and/or", "and/or" as used herein is intended to be inclusive of any one of the two or more items listed in association, and also to include any and all combinations of the items listed in association, including any two or more of the items listed in association, any more of the items listed in association, or all combinations of the items listed in association. It should be noted that when at least three items are connected by at least two conjunctive combinations selected from "and/or", "or" and/or ", it should be understood that in this application, the technical solutions unquestionably include the technical solutions all connected by" logical and ", and also unquestionably include the technical solutions all connected by" logical or ". For example, "A and/or B" includes A, B and A + B. For example, the embodiments of "a, and/or, B, and/or, C, and/or, D" include any of A, B, C, D (i.e., all embodiments using a "logical or" connection), any and all combinations of A, B, C, D, i.e., any two or any three of A, B, C, D, and four combinations of A, B, C, D (i.e., all embodiments using a "logical and" connection).
The present invention relates to "plural", etc., and indicates, unless otherwise specified, a number greater than 2 or equal to 2. For example, "one or more" means one or two or more.
As used herein, "a combination thereof," "any combination thereof," and the like, includes all suitable combinations of any two or more of the listed items.
In the present specification, the term "suitable" in "a suitable combination, a suitable manner," any suitable manner "and the like shall be construed to mean that the technical solution of the present invention can be implemented, the technical problem of the present invention can be solved, and the technical effect of the present invention can be achieved.
The terms "preferably", "better" and "suitable" are used herein only to describe preferred embodiments or examples, and it should be understood that the scope of the present invention is not limited by these terms.
In the present invention, "further", "still", "specifically", etc. are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of the present invention.
In the present invention, "optionally", "optional" and "optional" refer to the presence or absence, i.e., to any one selected from the two juxtapositions "present" or "absent". If multiple optional parts appear in one technical scheme, if no special description exists, and no contradiction or mutual constraint relation exists, each optional part is independent.
In the present invention, the terms "first", "second", "third", "fourth", etc. in the terms of "first aspect", "second aspect", "third aspect", "fourth aspect", etc. are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor as implying that importance or quantity indicating the technical feature being indicated. Also, "first," "second," "third," "fourth," etc. are used for non-exhaustive enumeration of description purposes only and should not be construed as a closed limitation to the number.
In the present invention, the technical features described in the open type include a closed technical solution composed of the listed features, and also include an open technical solution including the listed features.
In the present invention, where a range of values (i.e., a numerical range) is recited, unless otherwise specified, alternative distributions of values within the range are considered to be continuous, and include both the numerical endpoints of the range (i.e., the minimum and maximum values), and each numerical value between the numerical endpoints. Unless otherwise specified, when a numerical range refers to integers only within the numerical range, both endpoints of the numerical range and each integer between the two endpoints are included, and in this document, it is equivalent to reciting each integer directly, for example, t is an integer selected from 1 to 10, meaning t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. Further, when multiple range-describing features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, the ranges disclosed herein are to be understood to include any and all subranges subsumed therein.
The temperature parameter in the present invention is not particularly limited, and is allowed to be constant temperature treatment or to vary within a certain temperature range. It will be appreciated that the described thermostatic process allows the temperature to fluctuate within the accuracy of the instrument control. Allowing fluctuations in the temperature ranges of, for example, +/-5 deg.C, +/-4 deg.C, +/-3 deg.C, +/-2 deg.C and + -1 deg.C.
In the present invention,% (w/w) and wt% each represent a weight percentage,% (v/v) means a volume percentage, and% (w/v) means a mass volume percentage.
All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. The citation referred to herein is incorporated by reference in its entirety for all purposes unless otherwise in conflict with the present disclosure's objectives and/or technical solutions. Where a citation is referred to herein, the definition of a reference in the document, including features, terms, nouns, phrases, etc., that is relevant, is also incorporated by reference. In the present invention, when the citation is referred to, the cited examples and preferred embodiments of the related art features are also incorporated by reference into the present application, but the present invention is not limited to the embodiments. It should be understood that where a reference conflicts with the description herein, the application is controlling or adaptively modified in accordance with the description herein.
First aspect of the invention
The invention provides a serum-free culture medium for adenovirus production, which comprises 599.3mg/L-2226mg/L amino acid, 6.5mg/L-49.8mg/L vitamin, 5550.8mg/L-13845.3mg/L inorganic salt, 2150mg/L-5670mg/L glucose, 500mg/L-2000mg/L PF68, 1.2mg/L-7.5mg/L sodium hyaluronate and water;
wherein the vitamins comprise choline chloride, calcium pantothenate, nicotinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, and inositol.
The invention carries out comprehensive adjustment based on the traditional DMEM culture medium, comprises the selection of vitamin types, PF68 and sodium hyaluronate, and finally develops a culture medium which is protein-free, serum-free, hydrolysate-free, serum-free substitute and clear in chemical components. The culture medium is used for adenovirus production, is suitable for large-scale vaccine production, and is also suitable for culturing conventional host cells (such as HEK 293).
The culture medium provided by the invention can be added with phenol red or without phenol red according to needs.
In some embodiments of the invention, the choline chloride is at a concentration of 0.9mg/L to 7mg/L, the calcium pantothenate is at a concentration of 0.8mg/L to 8.2mg/L, the nicotinamide is at a concentration of 1.3mg/L to 7.8mg/L, the pyridoxine hydrochloride is at a concentration of 0.9mg/L to 8.4mg/L, the riboflavin is at a concentration of 0.2mg/L to 0.7mg/L, the thiamine hydrochloride is at a concentration of 3.7mg/L to 10mg/L, and the inositol is at a concentration of 1.7mg/L to 12 mg/L.
In some embodiments of the invention, the amino acid comprises one or more of glycine, arginine, L-cystine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, L-tyrosine, and valine.
In some embodiments of the invention, the glycine is at a concentration of 21mg/L to 48.3mg/L, the arginine is at a concentration of 28.8mg/L to 105.8mg/L, the L-cystine is at a concentration of 18.1mg/L to 79.4mg/L, the glutamine is at a concentration of 208.8mg/L to 735.8mg/L, the histidine is at a concentration of 29.4mg/L to 52.9mg/L, the isoleucine is at a concentration of 30.5mg/L to 132.3mg/L, the leucine is at a concentration of 33.5mg/L to 144.9mg/L, the lysine is at a concentration of 22.2mg/L to 184mg/L, the methionine is at a concentration of 21mg/L to 37.8mg/L, the phenylalanine is at a concentration of 26.2mg/L to 83.2mg/L, the concentration of the serine is 29.4mg/L-52.9mg/L, the concentration of the threonine is 46.5mg/L-119.7mg/L, the concentration of the tryptophan is 11.2mg/L-20.2mg/L, the concentration of the L-tyrosine is 32.8mg/L-131mg/L, and the concentration of the valine is 35.8mg/L-118.4 mg/L.
In some embodiments of the invention, the inorganic salt comprises one or more of calcium chloride, iron nitrate nonahydrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, and sodium dihydrogen phosphate monohydrate.
In some embodiments of the invention, the concentration of calcium chloride is 184.8mg/L-332.6mg/L, the concentration of iron nitrate nonahydrate is 0.1mg/L-2.1mg/L, the concentration of magnesium sulfate is 28.4mg/L-123.1mg/L, the concentration of potassium chloride is 180mg/L-504mg/L, the concentration of sodium bicarbonate is 2590mg/L-4662mg/L, the concentration of sodium chloride is 2480mg/L-8064mg/L, and the concentration of sodium dihydrogen phosphate monohydrate is 87.5mg/L-157.5 mg/L.
In some embodiments of the invention, the medium comprises 45mg/L to 48.3mg/L glycine, 100mg/L to 105.8mg/L arginine, 78.1mg/L to 79.4mg/L L-cystine, 600mg/L to 735.8mg/L glutamine, 49mg/L to 52.9mg/L histidine, 130mg/L to 132.3mg/L isoleucine, 140mg/L to 144.9mg/L leucine, 180mg/L to 184mg/L lysine, 35mg/L to 37.8mg/L methionine, 80mg/L to 83.2mg/L phenylalanine, 49mg/L to 52.9mg/L serine, 110mg/L to 119.7mg/L threonine, 19mg/L-20.2mg/L tryptophan, 125mg/L-131 mg/LL-tyrosine, 110mg/L-118.4mg/L valine, 6.5mg/L-7mg/L choline chloride, 6mg/L-7.5mg/L calcium pantothenate, 7.3mg/L-7.8mg/L nicotinamide, 7mg/L-8.4mg/L pyridoxine hydrochloride, 0.65mg/L-0.7mg/L riboflavin, 0.7mg/L-6.7mg/L thiamine hydrochloride, 11.5mg/L-12mg/L inositol, 310mg/L-332.6mg/L calcium chloride, 0.1mg/L-1.1mg/L ferric nitrate nonahydrate, 115mg/L-123.1mg/L magnesium sulfate, 500mg/L-504mg/L potassium chloride, 4300mg/L-4662mg/L sodium bicarbonate 7500mg/L-8064mg/L sodium chloride, 150mg/L-157.5mg/L monobasic sodium phosphate, 5200mg/L-5670mg/L glucose, 1900mg/L-2000mg/L PF68, 7mg/L-7.5mg/L sodium hyaluronate and water. In the culture medium environment, host cells (such as HEK293) can keep suspension growth, relieve the agglomeration phenomenon of the cells in the suspension culture process, facilitate cell proliferation and contribute to efficient adenovirus production.
Second aspect of the invention
The invention provides an adenovirus production method, which comprises the following steps: providing a host cell, placing the host cell in the culture medium of the first aspect, inoculating adenovirus, culturing, and harvesting adenovirus.
In some embodiments of the invention, the culturing is for a period of 72h to 96 h. For example 72h, 74h, 76h, 78h, 80h, 82h, 84h, 86h, 88h, 90h, 92h, 94h, 96 h. Preferably 96 h.
In some embodiments of the invention, the host cell is placed in the culture medium and cultured for 72h to 96h prior to inoculation with the adenovirus. For example 72h, 74h, 76h, 78h, 80h, 82h, 84h, 86h, 88h, 90h, 92h, 94h, 96 h. Preferably 96 h.
In some embodiments of the invention, the sinkThe density of the main cells in the medium was 2X 106cells/mL-6×106cells/mL. For example, 2X 106cells/mL、3×106cells/mL、4×106cells/mL、5×106cells/mL、6×106cells/mL。
In some embodiments of the invention, the adenovirus is administered at a concentration of 1MOI to 10 MOI.
In some embodiments of the invention, the host cell is HEK 293. HEK293 cells are easy to transfect and are a cell strain which is very commonly used for expressing and researching exogenous genes. Moreover, because of the translation and modification functions of 293 cells to proteins, the potency of the activity of the drug is better. For example, HEK293 cells are used as packaging cell lines for adenoviruses. Propagation of the virus occurs in living cells and replication of the virus in any non-living medium is not possible.
In the embodiment of the invention: PF68, manufacturer: sigma; the goods number is: k4894; pyridoxine hydrochloride, manufacturer: applichem; the goods number is: a8093; sodium hyaluronate, manufacturer: merck; the goods number is: s0780000.
Example 1 serum-free Medium for adenovirus production
HEK293 cells were placed in serum-free media for adenovirus production of different formulations as shown in Table 1 and Table 2, respectively, under the same culture conditions (37 ℃ C., 5% CO)2) The cell density, viability and cell state were measured every 24 hours in the lower culture from 0 hours, and the results are shown in tables 3 and 4. Meanwhile, DMEM medium was set as a control.
Table 1, serum-free medium for adenovirus production, units: mg/L
Table 2, serum-free medium for adenovirus production, unit: mg/L
Table 3 and Table 1 show the results of culturing serum-free Medium for adenovirus production
"cell dispersion" means that the cells are single cells, the cell surface is smooth, and the light transmittance is good; "slight clumping" means that 3-4 cells were seen under the microscope to adhere together; "Severe clumping" means that the cells in the culture vessel were hanging on a wall, small clumps were visible to the naked eye, and multiple cells were observed to adhere together under a microscope.
Table 4 and Table 2 show the culture results of serum-free Medium for adenovirus production
Example 2
HEK293 cells at 1X 106Inoculating cells/mL into the culture medium formula shown in Table 1, culturing for 48h, 72h and 96h respectively, and inoculating the virus, wherein the inoculation density comprises the following specific steps: the cells were transferred to a centrifuge tube, centrifuged at 1000rpm for 5min, the supernatant was discarded, and the cell density was adjusted to 5X 10 using formula 4 medium and DMEM medium of Table 16cells/mL, 1/1000 volumes of adenovirus stock solution were added, placed at 37 ℃ in 5% CO2Culturing in a shaking incubator at 125rpm, harvesting the virus liquid for about 96h, and detecting the cell density, the survival rate and the PF of the harvested liquidU, results are shown in Table 5. Meanwhile, DMEM medium was used as a control.
According to the experimental results, in the culture media of the formulas 1 to 4, the virus content per unit volume can be improved by more than 10 times compared with the virus production capacity of DMEM of the comparative example by preferably 72h and more preferably 96 h.
Table 5, summary of the results of example 2
Example 3
HEK293 cells were plated at 1X 106Inoculating cells/mL into the culture medium described in Table 1, culturing for 96h, transferring cells into a centrifuge tube, centrifuging at 1000rpm for 5min, discarding supernatant, and adjusting cell density to 2 × 10 with 4 different formula culture media described in Table 16cells/mL、3×106cells/mL、4×106cells/mL、5×106cells/mL、6×106cells/mL, 1/1000 volumes of adenovirus stock solution were added, placed at 37 ℃ in 5% CO2Culturing in a shaking incubator at 125rpm, harvesting the virus liquid for about 96 hours, and detecting the cell density, the survival rate and the PFU of the harvested liquid.
The results are shown in Table 6, and the cell density before inoculation is preferably 2X 106-6×106cells/mL, more preferably 5X 106cells/mL。
TABLE 6 cell Density optimization
Example 4
HEK293 cells at 1X 106cells/mL were inoculated into formula 4 medium described in Table 1 for 96h, cells were transferred to a centrifuge tube, centrifuged at 1000rpm for 5min, the supernatant was discarded, and the cell density was adjusted to 5X 10 using formula 4 medium described in example 16cells/mL, 1/1000 volumes of adenovirus stock solution were added, placed at 37 ℃ in 5% CO2And culturing in a shaking incubator at 125rpm, harvesting virus liquid for about 48h, 72h, 96h and 120h respectively, and detecting the cell density, the survival rate and the PFU of the harvested liquid.
The results are shown in Table 7, and the virus can be collected for 48h and 72h, preferably 96 h.
TABLE 7 optimal harvest time
Example 5 detection of infection efficiency
HEK293 cells were plated at 1X 106Inoculating cells/mL into the culture medium shown in Table 1 and Table 2, culturing for 96h, transferring cells into a centrifuge tube, centrifuging at 1000rpm for 5min, discarding supernatant, and adjusting cell density to 5 × 10 with the corresponding culture medium6cells/mL, 1/1000 volumes of adenovirus stock solution were added, placed at 37 ℃ in 5% CO2And culturing in a shaking incubator at 125rpm, harvesting virus liquid in 48h and 96h respectively, taking 20 mu L of the virus liquid, transferring the virus liquid to a glass slide, and observing the intensity and the number of fluorescence under a fluorescence microscope, wherein the stronger the intensity of the fluorescence, the more the number, the higher the transfection rate.
According to the cell growth results in tables 3 and 4, the proliferation rate of the HEK293 cells in the formula 4 is the fastest, the doubling time is about 24 hours, and the doubling time in the other formulas is more than 24 hours. To rule out the increase in fluorescence due to cell doubling, we chose 18h after inoculation for examination of transfection efficiency.
In addition, it takes 6 to 8 hours for the adenovirus particles to adhere to and enter the host cell until the mature infectious particles are finally released, and then it is faster, about only 4 to 6 hours, that is, the adenovirus particles can complete at least two rounds of propagation before the cells are doubled.
The results of fluorescence detection are shown in fig. 1 to fig. 9, and from the viewpoint of infection efficiency of adenovirus at 18 hours, the formulations in table 1 are all superior to the formulations in table 2, and the formulations in table 1 are preferably formulation 2 and formulation 3, and more preferably formulation 4.
The technical features of the embodiments and examples described above can be combined in any suitable manner, and for the sake of brevity, all possible combinations of the technical features of the embodiments and examples described above are not described, but should be considered within the scope of the present disclosure as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, so as to understand the technical solutions of the present invention specifically and in detail, but not to be understood as the limitation of the protection scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Furthermore, it should be understood that after reading the above teachings of the present invention, various changes or modifications may be made to the invention by those skilled in the art, and equivalents may be obtained and still fall within the scope of the present application. It should also be understood that the technical solutions provided by the present invention, which are obtained by logical analysis, reasoning or limited experiments, are within the scope of the present invention as set forth in the appended claims. Therefore, the protection scope of the present invention should be subject to the content of the appended claims, and the description and the drawings can be used for explaining the content of the claims.
Claims (10)
1. A serum-free culture medium for adenovirus production, which is characterized in that,
the culture medium comprises 599.3mg/L-2226mg/L amino acid, 6.5mg/L-49.8mg/L vitamin, 5550.8mg/L-13845.3mg/L inorganic salt, 2150mg/L-5670mg/L glucose, 500mg/L-2000mg/L PF68, 1.2mg/L-7.5mg/L sodium hyaluronate and water;
wherein the vitamins comprise choline chloride, calcium pantothenate, nicotinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, and inositol.
2. The serum-free medium for adenovirus production according to claim 1,
the concentration of the choline chloride is 0.9-7 mg/L, the concentration of the calcium pantothenate is 0.8-8.2 mg/L, the concentration of the nicotinamide is 1.3-7.8 mg/L, the concentration of the pyridoxine hydrochloride is 0.9-8.4 mg/L, the concentration of the riboflavin is 0.2-0.7 mg/L, the concentration of the thiamine hydrochloride is 3.7-10 mg/L, and the concentration of the inositol is 1.7-12 mg/L.
3. The serum-free medium for adenovirus production according to claim 1 or 2,
the amino acid comprises one or more of glycine, arginine, L-cystine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, L-tyrosine, and valine.
4. The serum-free medium for adenovirus production according to claim 3,
the concentration of the glycine is 21mg/L-48.3mg/L, the concentration of the arginine is 28.8mg/L-105.8mg/L, the concentration of the L-cystine is 18.1mg/L-79.4mg/L, the concentration of the glutamine is 208.8mg/L-735.8mg/L, the concentration of the histidine is 29.4mg/L-52.9mg/L, the concentration of the isoleucine is 30.5mg/L-132.3mg/L, the concentration of the leucine is 33.5mg/L-144.9mg/L, the concentration of the lysine is 22.2mg/L-184mg/L, the concentration of the methionine is 21mg/L-37.8mg/L, the concentration of the phenylalanine is 26.2mg/L-83.2mg/L, the concentration of the serine is 29.4mg/L-52.9mg/L, the concentration of the threonine is 46.5mg/L-119.7mg/L, the concentration of the tryptophan is 11.2mg/L-20.2mg/L, the concentration of the L-tyrosine is 32.8mg/L-131mg/L, and the concentration of the valine is 35.8mg/L-118.4 mg/L.
5. The serum-free medium for adenovirus production according to claim 1 or 2,
the inorganic salt comprises one or more of calcium chloride, ferric nitrate nonahydrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, and sodium dihydrogen phosphate monohydrate.
6. The serum-free medium for adenovirus production according to claim 5,
the concentration of the calcium chloride is 184.8-332.6 mg/L, the concentration of the ferric nitrate nonahydrate is 0.1-2.1 mg/L, the concentration of the magnesium sulfate is 28.4-123.1 mg/L, the concentration of the potassium chloride is 180-504 mg/L, the concentration of the sodium bicarbonate is 2590-4662 mg/L, the concentration of the sodium chloride is 2480-8064 mg/L, and the concentration of the sodium dihydrogen phosphate monohydrate is 87.5-157.5 mg/L.
7. The serum-free medium for adenovirus production according to any one of claims 1, 2, 4, and 6,
the culture medium contains 45mg/L-48.3mg/L glycine, 100mg/L-105.8mg/L arginine, 78.1mg/L-79.4mg/L L-cystine, 600mg/L-735.8mg/L glutamine, 49mg/L-52.9mg/L histidine, 130mg/L-132.3mg/L isoleucine, 140mg/L-144.9mg/L leucine, 180mg/L-184mg/L lysine, 35mg/L-37.8mg/L methionine, 80mg/L-83.2mg/L phenylalanine, 49mg/L-52.9mg/L serine, 110mg/L-119.7mg/L threonine, 19mg/L-20.2mg/L tryptophan, 125mg/L-131mg/L L-tyrosine, 110mg/L-118.4mg/L valine, 6.5mg/L-7mg/L choline chloride, 6mg/L-7.5mg/L calcium pantothenate, 7.3mg/L-7.8mg/L nicotinamide, 7mg/L-8.4mg/L pyridoxine hydrochloride, 0.65mg/L-0.7mg/L riboflavin, 0.7mg/L-6.7mg/L thiamine hydrochloride, 11.5mg/L-12mg/L inositol, 310mg/L-332.6mg/L calcium chloride, 0.1mg/L-1.1mg/L ferric nitrate nonahydrate, 115mg/L-123.1mg/L magnesium sulfate, 500mg/L-504mg/L potassium chloride, 4300mg/L-4662mg/L sodium bicarbonate 7500mg/L-8064mg/L sodium chloride, 150mg/L-157.5mg/L monobasic sodium phosphate, 5200mg/L-5670mg/L glucose, 1900mg/L-2000mg/L PF68, 7mg/L-7.5mg/L sodium hyaluronate and water.
8. A method for producing an adenovirus, comprising the steps of:
providing a host cell, placing the host cell in the culture medium of any one of claims 1 to 7, inoculating adenovirus, culturing, and harvesting the adenovirus.
9. The method for producing an adenovirus according to claim 8, wherein the culturing is carried out for 72 to 96 hours; or/and the host cell is placed in the culture medium to be cultured for 72h-96h and then is inoculated with the adenovirus.
10. The method for producing an adenovirus according to claim 8 or 9, wherein the density of the host cell in the culture medium is 2 x 106cells/mL-6×106cells/mL; or/and (c) the first and/or second,
the inoculation concentration of the adenovirus is 1MOI-10 MOI; or/and (c) the first and/or second,
the host cell is HEK293 cell.
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| CN115537378A (en) * | 2022-11-24 | 2022-12-30 | 天信和(苏州)生物科技有限公司 | Method for performing suspension culture on HEK293 cells and culture medium thereof |
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| CN111518768A (en) * | 2020-04-14 | 2020-08-11 | 四川百诺吉科技有限公司 | Low-serum culture medium suitable for LMH cell adherent culture and preparation method thereof |
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| CN115505571A (en) * | 2022-11-24 | 2022-12-23 | 上海倍谙基生物科技有限公司 | Serum-free additive suitable for suspension culture of LMH cells to produce avian adenovirus |
| CN115537378A (en) * | 2022-11-24 | 2022-12-30 | 天信和(苏州)生物科技有限公司 | Method for performing suspension culture on HEK293 cells and culture medium thereof |
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