CN113512521A

CN113512521A - Serum-free medium additive, serum-free medium and application thereof

Info

Publication number: CN113512521A
Application number: CN202110579641.7A
Authority: CN
Inventors: 施坤宁; 王星; 孙振华
Original assignee: Jiangsu Purecell Bio Medicine Technology Co ltd
Current assignee: Jiangsu Purecell Bio Medicine Technology Co ltd
Priority date: 2021-05-26
Filing date: 2021-05-26
Publication date: 2021-10-19
Anticipated expiration: 2041-05-26
Also published as: CN113512521B

Abstract

The invention belongs to the technical field of cell culture, and particularly relates to an additive of a serum-free culture medium, the serum-free culture medium and application thereof. The additive provided by the invention has the advantages of clear and synergistic mixture ratio of the components, good repeatability among different batches, high clinical safety, and capability of replacing animal-derived components such as serum and platelet lysate and meeting the in vitro culture requirements of immune cells such as PBMC and T cells. The serum-free culture medium prepared by the additive can realize high-efficiency culture of immune cells, and has high cell proliferation rate and low cell agglomeration rate.

Description

Serum-free medium additive, serum-free medium and application thereof

Technical Field

The invention belongs to the technical field of cell culture, and particularly relates to an additive of a serum-free culture medium, the serum-free culture medium and application thereof.

Background

Immune cells (immuno cells), commonly referred to as leukocytes, refer to cells involved in or associated with an immune response, including lymphocytes, dendritic cells, monocytes/macrophages, granulocytes, mast cells, and the like.

Peripheral Blood Mononuclear Cells (PBMC) refer to cells having a single nucleus in peripheral blood, including lymphocytes (T, B, NK), monocytes, and the like. T cells are one kind of lymphocytes derived from hematopoietic stem cells of human bone marrow, which migrate from the thymus into the thymus to differentiate and mature, and then further migrate into lymphoid tissues.

In recent years, since T cells have been used for experimental studies on the immune system or its related fields, it has been required to proliferate sufficient T cells. T cells have weak self-proliferation capacity, and the RPMI-1640 or DMEM medium commonly used for cell culture cannot provide nutrients necessary for the growth of the T cells. In order to promote the proliferation of T cells, animal-derived components are added into RPMI-1640 or DMEM medium, such as serum (human serum or fetal bovine serum) or Platelet Lysate (PL) as a base to support the in vitro amplification and subculture of T cells. However, the quality of serum varies greatly from batch to batch, requiring frequent screening for agents that may be detrimental to T cell expansion and survival; if human serum is adopted, on one hand, the price is high, and on the other hand, an exogenous reagent is needed to detect a possible new infectious factor. The first Serum Free Medium (SFM) was introduced to the market in 1965, based on which the elimination of serum from the cell culture medium to support robust expansion of T cells and clonal selection became a new direction for research in this field.

From the viewpoint that a new era of metabolic immunology is coming, and the interaction between immunity and metabolism is studied on the basis of metabolism and immune interrelation, it is clear that the interaction between lymphocyte metabolism and immunity is: how T cell metabolism affects T cell function; glucose, glutamine and serine are essential nutrients and work for T cell expansionEnergy is saved; metal ions (e.g. Ca)²⁺And Zn²⁺) Are important cofactors of proteins, are messengers of intracellular signals, and the like. Therefore, the development of a pure chemically defined serum-free cell culture medium to enable more immune cells to strongly expand and stably proliferate for a long time can achieve better clinical benefit.

Disclosure of Invention

Problems to be solved by the invention

In view of the problems of the prior art, for example, the cell culture gene components based on serum or Platelet Lysate (PL) are not clear, and the quality difference between different batches is large, thereby affecting the immune cell correlation experiment. Therefore, the invention provides the serum-free medium additive which does not contain components such as serum, platelet lysate and the like, is added into the serum-free medium, has clear sources and small batch-to-batch difference, and can realize high-efficiency culture of immune cells.

Means for solving the problems

The invention provides an additive of a serum-free culture medium, which comprises the following components: the first component containing basic metabolism substances is 4.2-20%, the second component containing cytokine substances is 75.5-94.4%, and the third component containing redox substances is 1.4-4.5%.

Preferably, the additive consists of: the first component containing basic metabolism substances is 4.2-20%, the second component containing cytokine substances is 75.5-94.4%, and the third component containing redox substances is 1.4-4.5%.

Preferably, the first component comprises one or a combination of more than two of dextran sodium sulfate, 1-thioglycerol, sodium pyruvate, ferric citrate amine, recombinant human insulin, ethanolamine, progesterone, adrenalone, thyroxine and estradiol.

Further preferably, the concentration of each component of the first component in the serum-free medium is as follows: 20-500mg/L of dextran sodium sulfate, 0.156-3.9mg/L of 1-thioglycerol, 22-550mg/L of sodium pyruvate, 2-50mg/L of ferric citrate amine, 0.8-20mg/L of recombinant human insulin, 0.2-5mg/L of ethanolamine, 0.126-3.15mg/L of progesterone, 0.04-1mg/L of adrenal ketone, 0.04-1mg/L of thyroxine, and 0.054-1.35mg/L of estradiol.

Preferably, the second component comprises one or a combination of two or more of human serum albumin, transferrin, interleukin 2, interleukin 15, LONGR3IGF-1, IGF-1 and bFGF.

Further preferably, the concentration of each component of the second component in the serum-free medium is as follows: 1-4g/L of human serum albumin, 1-25mg/L of transferrin, 100ng/mL of interleukin 210-.

Preferably, the third component comprises one or a combination of two or more of lipoic acid, tocopherol acetate, catalase, glutathione, superoxide enzyme, retinal, retinyl acetate, L-ascorbic acid and 2-mercaptoethanol.

Further preferably, the concentration of each component of the third component in the serum-free medium is as follows: 2.4-60mg/L lipoic acid, 0.2-5mg/L tocopherol acetate, 0.5-12.5mg/L catalase, 0.2-5mg/L glutathione, 0.5-12.5mg/L superoxide enzyme, 0.4-10mg/L retinal acetate, 10-100 mg/L-ascorbic acid and 0.78-7.8 mg/L2-mercaptoethanol.

The invention also provides a serum-free culture medium which comprises the additive.

Preferably, the serum-free culture medium further comprises a basic culture medium, and the mass ratio of the basic culture medium to the additive is (3-15): 1.

Preferably, the basal medium comprises amino acids, vitamins, inorganic salts, lipids, glucose, HEPES and phenol red.

Further preferably, the amino acid includes one or a combination of two or more of glycine, arginine, asparagine, aspartic acid, cystine, glutamic acid, glutamine, histidine, hydroxyproline, isoleucine, leucine, lysine hydrochloride, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

Further preferably, the vitamins include one or a combination of two or more of biotin, choline chloride, calcium D-pantothenate, folic acid, inositol, nicotinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, and p-aminobenzoic acid.

Further preferably, the inorganic salt comprises one or a combination of two or more of calcium nitrate tetrahydrate, magnesium sulfate, potassium chloride, disodium hydrogen phosphate and sodium chloride;

preferably, the lipid comprises one or a combination of two or more of arachidonic acid, cholesterol, DL-alpha-tocopheryl acetate, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid, stearic acid, tween 80 and F-68.

Still more preferably, the concentration of each component of said amino acids in said serum-free medium is as follows: 3.5-87.5mg/L of glycine, 65.28-1632mg/L of arginine, 12.64-316mg/L of asparagine, 6.66-166.5mg/L of aspartic acid, 14.8-370mg/L of cystine, 6.94-173.5mg/L of glutamic acid, 118.4-2960mg/L of glutamine, 11.4-285mg/L of histidine, 4-100mg/L of hydroxyproline, 20.48-512mg/L of isoleucine, 20.48-512mg/L of leucine, 22.5-562.5mg/L of lysine hydrochloride, 6.02-150.5mg/L of methionine, 9.6-240mg/L of phenylalanine, 6.3-157.5mg/L of proline, 8.1-202.5mg/L of serine, 13.52-338mg/L of threonine, 3.04-76mg/L of tryptophan, 11.2-280mg/L of tyrosine and 13.36-334mg/L of valine;

the concentrations of the individual components of the vitamins in the serum-free medium were as follows: 0.04-1mg/L of biotin, 0.8-20mg/L of choline chloride, 0.25-6.25mg/L of D-calcium pantothenate, 0.4-10mg/L of folic acid, 7.4-185mg/L of inositol, 0.4-10mg/L of nicotinamide, 0.4-10mg/L of pyridoxine hydrochloride, 0.06-1.5mg/L of riboflavin, 0.4-10mg/L of thiamine hydrochloride, 120.001-0.025 mg/L of vitamin B and 0.2-5mg/L of p-aminobenzoic acid;

the concentrations of the components of the inorganic salts in the serum-free medium were as follows: 20-500mg/L of calcium nitrate tetrahydrate, 9.768-244.2mg/L of magnesium sulfate, 80-2000mg/L of potassium chloride, 4000mg/L of disodium hydrogen phosphate, and 29675mg/L of sodium chloride 1187;

the concentrations of the individual components of the lipids in the serum-free medium were as follows: 0.0004-0.01mg/L of arachidonic acid, 0.044-1.1mg/L of cholesterol, 0.014-0.35mg/L of DL-alpha-tocopheryl acetate, 0.202-5.05mg/L of linoleic acid, 0.202-5.05mg/L of linolenic acid, 0.002-0.05mg/L of myristic acid, 0.202-5.05mg/L of oleic acid, 0.202-5.05mg/L of palmitic acid, 0.002-0.05mg/L of palmitoleic acid, 0.002-0.05mg/L of stearic acid, 800.44-11 mg/L of tween and 450mg/L of F-6818-sodium;

the concentration of the glucose in the serum-free medium is 1000-5000 mg/L;

the concentration of the HEPES in the serum-free medium is 1000-7000 mg/L;

the concentration of the phenol red in the serum-free culture medium is 1-10 mg/L.

The invention also provides the application of the additive or the serum-free culture medium in immune cell culture.

Preferably, the immune cells are selected from PBMCs or T cells.

ADVANTAGEOUS EFFECTS OF INVENTION

The serum-free medium additive provided by the invention has the advantages that the first component contains basic metabolism substances, the second component contains cytokine substances, the third component contains redox substances, the sources of the components are clear and the components are proportioned synergistically, the repeatability among different batches is good, the clinical safety is high, animal-derived components such as serum and platelet lysate can be replaced, and the in-vitro culture requirements of immune cells such as PBMC (peripheral blood mononuclear cell) and T cell are met.

The serum-free culture medium provided by the invention does not contain components such as serum, platelet lysate and the like, has clear sources and small batch-to-batch difference, can realize high-efficiency culture of immune cells, and has high cell proliferation rate and low cell agglomeration rate.

Drawings

FIG. 1 shows a comparison of cell morphology of PBMCs on day 1 on commercial and home-made serum-free media;

FIG. 2 shows a comparison of cell morphology of PBMCs on day 2 on commercial medium and home-made serum-free medium;

FIG. 3 shows a comparison of cell morphology of PBMCs on day 3 on commercial medium and home-made serum-free medium;

FIG. 4 shows a comparison of cell morphology of PBMCs on day 4 on commercial medium and home-made serum-free medium;

FIG. 5 shows a comparison of cell morphology of PBMCs on day 5 on commercial and home-made serum-free media;

FIG. 6 shows a comparison of cell morphology of PBMCs on day 6 on commercial and home-made serum-free media;

FIG. 7 shows a comparison of cell morphology of PBMCs at day 7 on commercial and home-made serum-free media;

FIG. 8 shows a comparison of cell morphology of PBMCs at day 8 on commercial and home-made serum-free media;

FIG. 9 shows a comparison of cell morphology of CD3+ T cells on day 6 on commercial medium and home-made serum-free medium;

FIG. 10 shows a comparison of cell morphology of CD3+ T cells on day 7 on commercial medium and home-made serum-free medium;

FIG. 11 shows a comparison of cell morphology of CD3+ T cells on day 8 in commercial and home-made serum-free media;

fig. 12 shows a comparison of cell morphology of CD3+ T cells on commercial medium and home-made serum-free medium at day 9;

FIG. 13 shows a comparison of cell morphology of CD3+ T cells on day 10 on commercial and home-made serum-free media;

FIG. 14 shows a comparison of cell morphology of CD3+ T cells on commercial medium and home-made serum-free medium at day 11;

fig. 15 shows a comparison of cell morphology of CD3+ T cells on day 12 on commercial medium and home-made serum-free medium;

fig. 16 shows a comparison of cell morphology of CD3+ T cells on day 13 on commercial medium and home-made serum-free medium;

fig. 17 shows a comparison of cell morphology of CD3+ T cells on commercial medium and home-made serum-free medium at day 14;

fig. 18 shows a comparison of cell morphology of CD3+ T cells on day 15 on commercial medium and home-made serum-free medium;

FIG. 19 shows the proliferation of PBMC cultured in commercial media and homemade serum-free media of examples 1, 2 and 3

FIG. 20 shows proliferation of CD3+ T cells in commercial media and home-made serum-free media in examples 1, 2, and 3.

FIG. 21 shows commercial culture medium culture T cell surface marker detection;

FIG. 22 shows home-made medium culture T cell surface marker detection.

Detailed Description

Various exemplary embodiments, features and aspects of the invention will be described in detail below. The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

Furthermore, in the following detailed description, numerous specific details are set forth in order to provide a better understanding of the present invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In other instances, methods, means, devices and steps which are well known to those skilled in the art have not been described in detail so as not to obscure the invention.

All units used in the specification are international standard units unless otherwise stated, and numerical values and numerical ranges appearing in the present invention should be understood to include systematic errors inevitable in industrial production.

In the present specification, "%" denotes mass% unless otherwise specified.

In the present specification, the meaning of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

In the present specification, reference to "some particular/preferred embodiments," "other particular/preferred embodiments," "embodiments," and the like, means that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various embodiments.

In the present specification, the numerical range represented by "numerical value a to numerical value B" means a range including the end point numerical value A, B.

In the present specification, the "normal temperature" and "room temperature" may be 10 to 40 ℃.

In addition, in the present specification, the "water" includes any available water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double distilled water, high purity water, and purified water.

First aspect

The invention provides an additive of a serum-free culture medium, which comprises the following components:

a first component containing a basal metabolic substance, a second component containing a cytokine-like substance, and a third component containing a redox-like substance;

wherein, the first component accounts for 4.2 to 20 percent, the second component accounts for 75.5 to 94.4 percent and the third component accounts for 1.4 to 4.5 percent by weight percentage.

In some preferred embodiments of the present invention, the additive is composed of: the first component accounts for 4.2-20 wt%, the second component accounts for 75.5-94.4 wt%, and the third component accounts for 1.4-4.5 wt%.

The first component of the serum-free medium additive provided by the invention can provide various substances required by metabolism for immune cells, and improve the metabolic activity of the cells; the second component can provide various factors required by proliferation and growth for immune cells and promote cell proliferation; the third component can provide the immune cells with a redox system required for maintaining the cellular morphology and biological characteristics of the immune cells, and prevent cell deformation and loss of immune cell characteristics. The components are proportioned according to specific mass, have clear sources, are cooperated and act together, have good repeatability among different batches and high clinical safety, can replace animal-derived components such as serum, platelet lysate and the like, and meet the in vitro culture requirements of immune cells such as PBMC, T cells and the like.

A first component

The first component contains a basal metabolic class of substances, which provide the classes of substances required for metabolism for immune cell proliferation. Specifically, the content of the first component is 4.2% to 20%, preferably 7.9% to 20%, by weight, and exemplarily, the content of the first component may be 4.2%, 7.9%, 12.5%, 15%, 17.5%, 19.5%, 20%. When the content of the first component is within the above range, the metabolism of immune cells can be promoted, and the cell proliferation rate can be increased.

In some specific embodiments of the invention, the first component comprises one or a combination of two or more of dextran sulfate sodium, 1-thioglycerol, sodium pyruvate, ferric citrate amine, recombinant human insulin, ethanolamine, progesterone, adrenal ketone, thyroxine, and estradiol, preferably including all of the above components.

Wherein, the dextran sodium sulfate is an anti-cell caking agent in the immune cell proliferation process; 1-thioglycerol is an important component for regulating cell growth metabolism; the sodium pyruvate plays a role of replacing a carbon source in a culture medium and participates in cell nutrition metabolism; ferric citrate amine plays a role in replacing a nitrogen source in a culture medium and participates in cell nutrition metabolism; the recombinant insulin simultaneously promotes the synthesis of glycogen, fat and protein, and is indispensable in cell growth and metabolism; ethanolamine is used as a synthetic precursor of cephalin and can replace lipids in serum; progesterone, adrenalone, thyroxine and estradiol are used as hormones, can regulate the metabolism of three nutrients such as protein, sugar and fat, and provide energy for the growth of immune cells.

Specifically, the concentrations of the components of the first component in serum-free medium were as follows: the concentration of dextran sodium sulfate is 20-500mg/L, preferably 100 mg/L; the concentration of the 1-thioglycerol is 0.156-3.9mg/L, and preferably 0.78 mg/L; the concentration of the sodium pyruvate is 22-550mg/L, preferably 110 mg/L; the concentration of the citric acid ferrylamine is 2-50mg/L, preferably 10 mg/L; the concentration of the recombinant human insulin is 0.8-20mg/L, preferably 4 mg/L; the concentration of the ethanolamine is 0.2-5mg/L, preferably 1 mg/L; the concentration of the progesterone is 0.126-3.15mg/L, preferably 0.63 mg/L; the concentration of the adrenalone is 0.04-1mg/L, preferably 0.2 mg/L; the concentration of thyroxine is 0.04-1mg/L, preferably 0.2 mg/L; the concentration of estradiol is 0.054-1.35mg/L, preferably 0.27 mg/L.

When the content of each component of the first component meets the requirement, the growth and metabolism of immune cells can be met; when the content of a certain component does not meet the above requirements, the growth metabolism of immune cells is hindered.

A second component

The second component contains cytokine-like substances, such as interleukins and growth factors, which promote immune cell proliferation. Specifically, the content of the second component is 75.5 to 94.4 percent, preferably 87.6 to 94.4 percent by weight; illustratively, the second component may be present in an amount of 75.5%, 87.6%, 89.5%, 91.5%, 93.5%, 94.4%, etc. When the content of the second component is within the above range, the metabolism of immune cells can be promoted, and the cell proliferation rate can be increased.

In some specific embodiments of the invention, the second component comprises one or a combination of two or more of human serum albumin, transferrin, interleukin 2, interleukin 15, LONGR3IGF-1, IGF-1 and bFGF, preferably all of the above components.

Wherein, Human Serum Albumin (HSA) is an important nutrient for cell growth and metabolism, and amino acid generated by decomposition of HSA can be used for cell synthesis of tissue protein; in addition, the human serum albumin can also provide proper medium osmotic pressure to meet the osmotic pressure balance of cell growth. Transferrin can regulate the balance of iron ions in cells, and realize the maintenance of cell growth and proliferation. The interleukin refers to a lymphokine interacting between leukocytes or immune cells, and plays an important role in information transmission, immune cell activation and regulation, T, B cell activation, proliferation and differentiation mediation and the like; wherein, the interleukin 2 is a T cell growth factor which can stimulate T cells to enter a cell division cycle and improve the proliferation rate of the T cells; in addition to having biological effects similar to those of interleukin 2, interleukin 15 is also capable of stimulating and maintaining the immune response of T cells. The growth factor is a cytokine with cell growth stimulating activity, and the human long R3 insulin-like growth factor (LONGR3IGF-1) can promote division, migration and differentiation of PBMC and T cells, and regulate metabolism of sugar and fat. IGF-1(insulin like growth factor-1) is a group of polypeptides with growth promoting effect. bfgf (basic fibroblast growth factor), which is a basic fibroblast growth factor, is a polypeptide that transmits a developmental signal to promote cell division, and can promote cell proliferation.

Specifically, the concentrations of the components of the second component in serum-free medium were as follows: the concentration of the human serum albumin is 1-4g/L, preferably 2.5 g/L; the concentration of transferrin is 1-25mg/L, preferably 5 mg/L; the concentration of interleukin 2 is 10-100ng/mL, preferably 50 ng/mL; the concentration of interleukin 15 is 10-100ng/mL, preferably 50 ng/mL; the concentration of LONGR3IGF-1 is 5-20ng/mL, preferably 10 ng/mL; the concentration of IGF-1 is 5-20ng/mL, preferably 10 ng/mL; the concentration of bFGF is 10-100ng/mL, preferably 50 ng/mL. When the content of each component of the second component meets the requirement, the immune cells can keep higher growth activity; when the content of a certain component does not meet the requirement, the growth and metabolism of immune cells are hindered.

Third component

The third component contains redox substances, and specifically, the content of the third component is 1.4-4.5% by weight, preferably 4.0-4.5% by weight; illustratively, the third component may be present in an amount of 1.4%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, etc. When the content of the third component is within the above range, the metabolism of immune cells can be promoted, and the cell proliferation rate can be increased.

In some specific embodiments of the invention, the third component comprises one or a combination of two or more of lipoic acid, tocopherol acetate, catalase, glutathione, superoxide enzyme, retinal acetate, L-ascorbic acid, and 2-mercaptoethanol, preferably all of the above.

Wherein, the lipoic acid belongs to a class of compounds in B vitamins, is a coenzyme existing in mitochondria, and can eliminate free radicals which cause accelerated aging and pathogenicity; tocopherol and tocopherol acetate are vitamin E indispensable to cells; the catalase can remove the metabolic waste hydrogen peroxide of cells, plays a role in detoxifying the cells, and has the functions of an antioxidant and resisting apoptosis; glutathione has antioxidant effect; the superoxide enzyme has the function of clearing oxygen free radicals, and can reduce the toxicity of the accumulation of the oxygen free radicals to cells; l-ascorbic acid is a high-stability vitamin C derivative, has strong oxidation resistance and anti-apoptosis effect, and participates in the growth and proliferation of stem cells and the like; 2-mercaptoethanol is a strong reducing agent that neutralizes oxygen radicals generated during cell growth and division to reduce cell damage.

Specifically, the concentrations of the components of the third component in the serum-free medium were as follows: the concentration of the lipoic acid is 2.4-60mg/L, and the preferable concentration is 12 mg/L; the concentration of the tocopherol is 0.2-5mg/L, preferably 1 mg/L; the concentration of the tocopherol acetate is 0.2-5mg/L, preferably 1 mg/L; the concentration of catalase is 0.5-12.5mg/L, preferably 2.5 mg/L; the concentration of the glutathione is 0.2-5mg/L, preferably 1 mg/L; the concentration of the superoxide enzyme is 0.5-12.5mg/L, preferably 2.5 mg/L; the concentration of retinene is 0.4-10mg/L, preferably 2 mg/L; the concentration of retinyl acetate is 0.4-10mg/L, preferably 2 mg/L; the concentration of the L-ascorbic acid is 10-100mg/L, preferably 100 mg/L; the concentration of 2-mercaptoethanol is 0.78-7.8mg/L, preferably 4.3 mg/L. When the content of each component of the third component meets the requirement, the immune cells can keep higher growth activity; when the content of a certain component does not meet the requirement, the growth and metabolism of immune cells are hindered.

Second aspect of the invention

The invention provides a serum-free culture medium, which comprisesFirst aspectProvided is an additive.

In some specific embodiments of the invention, the serum-free medium comprises, in addition to the above-described additives, a basal medium containing basic nutrients required for the proliferation of immune cells; wherein the mass ratio of the basic culture medium to the additive is (3-15) to 1, preferably 6: 1; illustratively, the mass ratio of the basal medium to the additives can be 3:1, 6:1, 8:1, 10:1, 12:1, 14:1, 15:1, and the like.

In some specific embodiments of the invention, the basic nutrients of the basal medium comprise amino acids, vitamins, inorganic salts, lipids, glucose, HEPES, phenol red, and the like.

The serum-free medium contains basal metabolism substances, cytokine substances and redox substances in additives, has clear sources of the components, does not contain components such as serum and platelet lysate, is cooperated with basic nutrients of a basal medium, has high clinical safety, can realize high-efficiency culture of immune cells (such as PBMC, T cells and the like), and has high cell proliferation rate and low cell aggregation rate.

Amino acids

The amino acid is used as an energy source of immune cells, participates in the metabolic process of substances such as proteins, nucleic acids and lipids of the immune cells, and maintains the growth and metabolism of the cells.

In some specific embodiments of the invention, the amino acid comprises one or a combination of two or more of glycine, arginine, asparagine, aspartic acid, cystine, glutamic acid, glutamine, histidine, hydroxyproline, isoleucine, leucine, lysine hydrochloride, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and alanine; the respective components including the above amino acids are preferred.

In some specific embodiments of the invention, the concentrations of the components of the amino acid in the serum-free medium are as follows: 3.5-87.5mg/L of glycine, preferably 10 mg/L; arginine 65.28-1632mg/L, preferably 200 mg/L; 12.64-316mg/L of asparagine, preferably 50 mg/L; aspartic acid 6.66-166.5mg/L, preferably 20 mg/L; cystine 14.8-370mg/L, preferably 65 mg/L; glutamic acid 6.94-173.5mg/L, preferably 20 mg/L; glutamine 118.4-2960mg/L, preferably 300 mg/L; histidine 11.4-285mg/L, preferably 15 mg/L; hydroxyproline 4-100mg/L, preferably 20 mg/L; isoleucine 20.48-512mg/L, preferably 50 mg/L; leucine in 20.48-512mg/L, preferably 50 mg/L; lysine hydrochloride 22.5-562.5mg/L, preferably 40 mg/L; methionine 6.02-150.5mg/L, preferably 15 mg/L; phenylalanine 9.6-240mg/L, preferably 15 mg/L; proline 6.3-157.5mg/L, preferably 20 mg/L; serine 8.1-202.5mg/L, preferably 30 mg/L; threonine 13.52-338mg/L, preferably 20 mg/L; 3.04-76mg/L tryptophan, preferably 5mg/L tryptophan; 11.2-280mg/L tyrosine, preferably 29mg/L tyrosine; valine is 13.36-334mg/L, preferably 20 mg/L.

Vitamin preparation

The vitamins can promote immune cell proliferation. In some specific embodiments of the invention, the vitamins include biotin, choline chloride, calcium D-pantothenate, folic acid, inositol, niacinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamins, and p-aminobenzoic acid; the components comprising the above vitamins are preferred.

In some specific embodiments of the invention, the concentration of the components of the vitamins in the serum-free medium is as follows: biotin 0.04-1mg/L, preferably 0.2 mg/L; 0.8-20mg/L of choline chloride, preferably 3 mg/L; 0.25-6.25mg/L, preferably 0.25mg/L, of calcium D-pantothenate; folic acid 0.4-10mg/L, preferably 1 mg/L; inositol 7.4-185mg/L, preferably 35 mg/L; nicotinamide 0.4-10mg/L, preferably 1 mg/L; pyridoxine hydrochloride in an amount of 0.4-10mg/L, preferably 1 mg/L; riboflavin is 0.06-1.5mg/L, preferably 0.2 mg/L; thiamine hydrochloride 0.4-10mg/L, preferably 1 mg/L; vitamin B120.001-0.025 mg/L, preferably 0.005 mg/L; 0.2-5mg/L, preferably 1mg/L of p-aminobenzoic acid.

Inorganic salt

The inorganic salt is beneficial to maintaining the osmotic pressure balance of the culture medium, participates in important physiological metabolic activities of cells, and has important significance for maintaining the stability of the cell state. In some specific embodiments of the invention, the inorganic salts include calcium nitrate tetrahydrate, magnesium sulfate, potassium chloride, disodium hydrogen phosphate, and sodium chloride; the respective components including the above inorganic salts are preferable.

In some specific embodiments of the invention, the concentrations of the components of the inorganic salts in the serum-free medium are as follows: 20-500mg/L of calcium nitrate tetrahydrate, preferably 100 mg/L; magnesium sulfate 9.768-244.2mg/L, preferably 48.84 mg/L; 80-2000mg/L potassium chloride, preferably 400 mg/L; disodium hydrogen phosphate 160-4000mg/L, preferably 800 mg/L; sodium chloride 1187-29675mg/L, preferably 5850 mg/L.

Lipids

The lipid and amino acid have similar effects, and can be used as energy source of immunocyte to participate in metabolism process of protein, nucleic acid, etc. of immunocyte for maintaining growth and metabolism of cells. In some specific embodiments of the invention, the lipid comprises one or a combination of two or more of arachidonic acid, cholesterol, DL-alpha-tocopheryl acetate, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid, stearic acid, tween 80 and F-68; preferably comprising the components of the above lipids.

In some specific embodiments of the invention, the concentrations of the components of the lipid in serum-free medium are as follows: arachidonic acid 0.0004-0.01mg/L, preferably 0.002 mg/L; cholesterol 0.044-1.1mg/L, preferably 0.22 mg/L; DL-alpha-tocopherol acetate 0.014-0.35mg/L, preferably 0.07 mg/L; linoleic acid 0.002-0.05mg/L, preferably 0.01 mg/L; linolenic acid 0.002-0.05mg/L, preferably 0.01 mg/L; myristic acid 0.002-0.05mg/L, preferably 0.01 mg/L; oleic acid 0.002-0.05mg/L, preferably 0.01 mg/L; palmitic acid 0.002-0.05mg/L, preferably 0.01 mg/L; palmitoleic acid 0.002-0.05mg/L, preferably 0.01 mg/L; stearic acid 0.002-0.05mg/L, preferably 0.01 mg/L; tween 800.44-11 mg/L, preferably 2.2mg/L, F-6818-450 mg/L, preferably 90 mg/L.

Glucose

In some specific embodiments of the invention, the concentration of glucose in serum-free medium is 1000-5000mg/L, preferably 2000 mg/L.

HEPES

In some specific embodiments of the invention, the concentration of HEPES in serum-free medium is 1000-7000mg/L, preferably 5958 mg/L.

Phenol Red

In some specific embodiments of the invention, the concentration of phenol red in serum-free medium is 1-10mg/L, preferably 5 mg/L.

Third aspect of the invention

The invention provides a method for preparingFirst aspectAdditives provided or according toSecond aspect of the inventionThe application of the serum-free culture medium in immune cell culture is provided.

The additive provided by the invention has the advantages that the first component contains a basic metabolism substance, the second component contains a cytokine substance, the third component contains a redox substance, the sources of the components are clear and are matched synergistically, animal-derived components such as serum and platelet lysate can be replaced, the increment rate of cells can be improved, and the agglomeration rate of the cells can be reduced.

The serum-free culture medium provided by the invention contains the additive, does not contain components such as serum, platelet lysate and the like, has clear sources and small batch-to-batch difference, can realize high-efficiency culture of immune cells, and has high cell proliferation rate and low cell agglomeration rate.

In some specific embodiments of the invention, the immune cells are selected from PBMCs or T cells.

Examples

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1

The following table shows the components of the basal medium and the working concentrations in serum-free medium.

TABLE 1

TABLE 2

TABLE 3

TABLE 4

TABLE 5

The following table shows the components and working concentrations of the additives in serum-free medium:

TABLE 6-1

TABLE 7-1

TABLE 8-1

In the serum-free medium, the mass ratio of the basic medium to the additive is 6:1, wherein in the additive, the first component is 7.9%, the second component is 87.6% and the third component is 4.5% in percentage by weight.

Example 2

The basic medium in this example has the same composition and working concentration as in example 1.

TABLE 6-2

TABLE 7-2

TABLE 8-2

In the serum-free medium, the mass ratio of the basic medium to the additive is 15:1, wherein in the additive, the first component is 4.2%, the second component is 94.4% and the third component is 1.4% in percentage by weight.

Example 3

tables 6 to 3

Tables 7 to 3

Tables 8 to 3

In the serum-free medium, the mass ratio of the basic medium to the additive is 3:1, wherein in the additive, the first component is 20%, the second component is 75.5% and the third component is 4.5% in percentage by weight.

Evaluation of Effect

1PBMC culture

1.1 isolation of mononuclear cells (PBMC) from peripheral blood

The density of various blood cells in peripheral blood varies, and the separation of mononuclear cells in blood is usually performed by density gradient centrifugation. The Ficoll-diatrizoate (F-H) layered liquid is the most commonly used separation liquid for density gradient centrifugation, and lymphocyte layered liquid (Ficoll) is used for density gradient centrifugation to distribute cell populations with a certain specific gravity according to corresponding density gradients, so that various blood cells are separated.

(1) Blood samples were prepared and 60mL of whole blood was transferred to three 50mL centrifuge tubes, 20mL per tube. An equal amount of D-PBS was added to 20mL of whole blood to make up to 40mL, and the mixture was inverted and mixed to obtain a diluted blood cell solution. 4 new 50mL centrifuge tubes were taken, 15mL of Ficoll solution was added to each tube, and 30mL of diluted blood cell solution was carefully added to each tube of Ficoll solution using an electrokinetic pipette (care was taken to establish stratification). Centrifuging at 20 deg.C for 20min at 800g, and increasing 1 and decreasing 0. After centrifugation, the centrifugal tube can be seen to be divided into four layers, wherein the first layer is yellow serum solution, the third layer is white ficoll solution, and a thin white film layer is arranged between the first layer and the third layer and is the required white film layer.

(2) Carefully sucking the tunica albuginea layer with an electric pipette, transferring to a new 50mL centrifuge tube, adding washing solution (D-PBS + 10% human serum albumin) to 40mL, reversing, mixing, centrifuging at 20 deg.C 1500rpm (or 491g) for 10min, discarding the supernatant, adding 40mL washing solution, resuspending the cells, centrifuging at 20 deg.C 1000rpm (or 491g) for 10min, discarding the supernatant (a small amount of red blood cells can be seen)Precipitation and PBMC precipitation), adding 40mL sorting buffer, resuspending cell precipitation, filtering with 70 μm filter, counting, centrifuging at 20 deg.C 800rpm (or 491g) for 8min to obtain 1 × 10 per mL of peripheral blood⁶～2×10⁶PBMC。

1.2 culture of PBMC

PBMC were cultured in the Serum-Free Medium of the present invention and a commercially available Lymphocyte Serum-Free Medium KBM 502, and the difference between PBMC cultured in the two groups was compared.

1.3 results of the experiment

1.3.1 cell morphology

FIGS. 1 to 8 show the observation results of cell morphology under 4-fold mirror (4X) and 10-fold mirror (10X) after PBMC were cultured on a commercially available medium and a self-prepared medium of example 1 of the present invention, respectively.

As can be seen from FIGS. 1 to 8, the PBMC cultured in the home-made culture medium of the present invention have a small and low cell aggregation rate.

1.3.2 cell proliferation

Table 9 and FIG. 19 show the cell proliferation of PBMCs cultured in commercial media and the home-made serum-free media of examples 1-3 of the present invention, respectively.

TABLE 9 proliferation of PBMC in Serum-Free Medium and in Serum-Free Medium KBM 502 Medium

As can be seen from Table 9 and FIG. 19, the PBMC cultured in the home-made medium according to the composition ratios of examples 1 to 3 proliferated faster.

2 CD3+ T cell culture

2.1 sorting CD3+ T cells

Magnetic beads sorted CD3+ T cells. The method for separating cells by the immunomagnetic bead method is based on the fact that cell surface antigen can be combined with specific monoclonal antibody connected with magnetic beads, in an external magnetic field, cells connected with the magnetic beads through the antibody are adsorbed and retained in the magnetic field, and cells without the surface antigen have no magnetism because the cells cannot be combined with the specific monoclonal antibody connected with the magnetic beads and do not stay in the magnetic field, so that the cells are separated.

(1) Sorting buffer (gentle: D-PBS + 0.5% BSA +2mM/L EDTA) was added to the cell pellet at a ratio of 80. mu.L/107 cells, and the cell pellet was resuspended.

(2) Then, CD3 magnetic beads were added at a ratio of 20. mu.L/107 cells, and after being mixed well by pipetting, the mixture was incubated at 4 ℃ for 15min without disturbance.

(3) The magnetic bead-cell mixture was removed, and the mixture was added to a sorting buffer at a ratio of 2mL/107cells, washed by mixing, and centrifuged at 1500rpm (or 491g) at 4 ℃ for 10 min.

(4) Add sorting buffer at 500. mu.L/108 cells and resuspend the cell pellet (500. mu.L in 1X108cells, 1mL in 2X108cells, 1.5mL in 3X108cells, and so on).

(5) Preparing a separation frame and a large separation column (LS), clamping the LS by using tweezers, and fixing the LS on a magnet frame; simultaneously, 3 15mL centrifuge tubes are prepared and respectively filled with: sorting buffer (tube A), CD 3-cytosol (tube B), CD3+ cytosol (tube C).

(6) The column was washed with 3mL of sorting buffer and the sorting buffer was connected to the a-tube.

(7) 1mL of cell-magnetic bead suspension was added and the column was washed with 3mL of sorting buffer (a new liquid was added each time no liquid remained) three times in total and collected in tube B to give CD 3-cells (stored at 4 ℃ for quality control).

(8) 5mL of sorting buffer was added and the inner plug of the column was washed slightly vigorously and collected in C-tube to give CD3+ cells. The sample was taken to count the amount of cells in 5 mL.

2.2 culture of CD3+ T cells

(1) Cells were resuspended in 10-20mL of medium (no IL-2) and factor 1000x A (IFN-. gamma.) was added. Adjusting 2X10^ 6/mL. The overnight coated T75 vial was removed, the coating was decanted, washed twice with D-PBS, and the cell suspension was inoculated into a T75 vial, shaken and observed under a microscope: the cell density is high, the fish egg is bright, the culture medium is clean and has no impurity cell, the bottle cap is unscrewed and put into the incubator for culture, and simultaneously CO in the incubator is added₂Adjusted to 6% (increase of CO in bottle)₂Concentration, favoring cell growth).

(2) T cells were cultured in a commercially available Lymphocyte Serum-Free Medium KBM 502.

2.3 results of the experiment

2.3.1 cell morphology

FIGS. 9-18 show the observation of cell morphology of CD3+ T cells after culturing on commercial 502 media and home-made serum-free media of example 1 of the present invention under 4-fold (4X) and 10-fold (10X) mirrors, respectively.

As can be seen from FIGS. 9 to 18, the CD3+ T cells cultured in the home-made culture medium of the present invention have a low clumping rate.

2.3.2 cell proliferation

Table 10 and FIG. 20 show the proliferation of CD3+ T cells after culturing in commercial media and in serum-free media prepared in accordance with examples 1-3 of the present invention, respectively.

TABLE 10 proliferation of CD3+ T cells in Serum-Free Medium and in Serum-Free Medium KBM 502 Medium

As can be seen from Table 10 and FIG. 20, the proliferation of CD3+ T cells cultured in the in-house culture medium according to the composition ratios of examples 1 to 3 of the present invention was fast.

2.3.3T cell surface marker evaluation

(1) Cell preparation: the cells are filtered by a 70-micron screen into a 50mL centrifuge tube, centrifuged for 5min at 300g, the supernatant is discarded, 30-40mL PBS is added, mixed well, and centrifuged for 5min at 300 g. Discard the supernatant, add PBS, adjust the cell concentration to about 5x10^ 6/mL.

(2) Sample preparation: 100. mu.L of the cell suspension was added to each flow tube.

(3) Incubation of cells with antibodies: the corresponding fluorescent antibody was added to the cell suspension and incubated at room temperature in the dark for 15 min.

(4) Adding a proper amount of PBS into each sample after incubation is finished, centrifuging for 5-10min at the temperature of 20 ℃ at 300g, and removing supernatant to leave cell precipitates; an appropriate amount of PBS was added to each sample to resuspend the cells, and the cells were centrifuged at 300g for 5-10min at 20 ℃.

(5) Adding a proper volume of PBS solution to prepare cell suspension (machine detection, at least 10000 cells are collected in each sample), and detecting the expression condition of each molecule by using a flow cytometer.

The fluorescent antibodies include anti-CD 4 antibody, anti-CD 3 antibody, and anti-CD 8a antibody.

The results of the examination of stool surface markers by flow assay showed no significant difference in the ratio of surface markers between T cells cultured in the commercial medium and the home-made medium, as shown in fig. 21 and 22.

Industrial applicability

The serum-free medium additive, the serum-free medium and the application thereof provided by the invention can be applied in industry.

The above examples of the present invention are merely examples for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. An additive for a serum-free medium, which is characterized by comprising the following components: the first component containing basic metabolism substances is 4.2-20%, the second component containing cytokine substances is 75.5-94.4%, and the third component containing redox substances is 1.4-4.5%;

2. The additive of claim 1, wherein the first component comprises one or a combination of two or more of sodium dextran sulfate, 1-thioglycerol, sodium pyruvate, ferric citrate, recombinant human insulin, ethanolamine, progesterone, adrenalone, thyroxine, and estradiol;

preferably, the concentrations of the components of the first component in the serum-free medium are as follows: 20-500mg/L of dextran sodium sulfate, 0.156-3.9mg/L of 1-thioglycerol, 22-550mg/L of sodium pyruvate, 2-50mg/L of ferric citrate amine, 0.8-20mg/L of recombinant human insulin, 0.2-5mg/L of ethanolamine, 0.126-3.15mg/L of progesterone, 0.04-1mg/L of adrenal ketone, 0.04-1mg/L of thyroxine, and 0.054-1.35mg/L of estradiol.

3. The supplement of claim 1 or 2, wherein the second component comprises one or a combination of two or more of human serum albumin, transferrin, interleukin 2, interleukin 15, LONGR3IGF-1, and bFGF;

preferably, the concentrations of the components of the second component in the serum-free medium are as follows: 1-4g/L of human serum albumin, 1-25mg/L of transferrin, 100ng/mL of interleukin 210-.

4. The additive of any one of claims 1-3, wherein the third component comprises one or a combination of two or more of lipoic acid, tocopherol acetate, catalase, glutathione, superoxide enzyme, retinal acetate, L-ascorbic acid, and 2-mercaptoethanol;

preferably, the concentration of each component of the third component in the serum-free medium is as follows: 2.4-60mg/L lipoic acid, 0.2-5mg/L tocopherol acetate, 0.5-12.5mg/L catalase, 0.2-5mg/L glutathione, 0.5-12.5mg/L superoxide enzyme, 0.4-10mg/L retinal acetate, 10-100 mg/L-ascorbic acid and 0.78-7.8 mg/L2-mercaptoethanol.

5. A serum-free medium comprising the additive according to any one of claims 1-4.

6. The serum-free medium according to claim 5, wherein the serum-free medium further comprises a basal medium, and the mass ratio of the basal medium to the additive is (3-15): 1.

7. The serum-free medium according to claim 5 or 6, wherein the basal medium comprises amino acids, vitamins, inorganic salts, lipids, glucose, HEPES and phenol red;

preferably, the amino acid comprises one or a combination of more than two of glycine, arginine, asparagine, aspartic acid, cystine, glutamic acid, glutamine, histidine, hydroxyproline, isoleucine, leucine, lysine hydrochloride, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine;

preferably, the vitamins include one or a combination of two or more of biotin, choline chloride, calcium D-pantothenate, folic acid, inositol, nicotinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, and p-aminobenzoic acid;

preferably, the inorganic salt comprises one or a combination of more than two of calcium nitrate tetrahydrate, magnesium sulfate, potassium chloride, disodium hydrogen phosphate and sodium chloride;

8. The serum-free medium according to claim 7, wherein the concentration of each component of the amino acids in the serum-free medium is as follows: 3.5-87.5mg/L of glycine, 65.28-1632mg/L of arginine, 12.64-316mg/L of asparagine, 6.66-166.5mg/L of aspartic acid, 14.8-370mg/L of cystine, 6.94-173.5mg/L of glutamic acid, 118.4-2960mg/L of glutamine, 11.4-285mg/L of histidine, 4-100mg/L of hydroxyproline, 20.48-512mg/L of isoleucine, 20.48-512mg/L of leucine, 22.5-562.5mg/L of lysine hydrochloride, 6.02-150.5mg/L of methionine, 9.6-240mg/L of phenylalanine, 6.3-157.5mg/L of proline, 8.1-202.5mg/L of serine, 13.52-338mg/L of threonine, 3.04-76mg/L of tryptophan, 11.2-280mg/L of tyrosine and 13.36-334mg/L of valine;

the concentrations of the individual components of the lipids in the serum-free medium were as follows: 0.0004-0.01mg/L of arachidonic acid, 0.044-1.1mg/L of cholesterol, 0.014-0.35mg/L of DL-alpha-tocopheryl acetate, 0.002-0.05mg/L of linoleic acid, 0.002-0.05mg/L of linolenic acid, 0.002-0.05mg/L of myristic acid, 0.002-0.05mg/L of oleic acid, 0.002-0.05mg/L of palmitic acid, 0.002-0.05mg/L of palmitoleic acid, 0.002-0.05mg/L of stearic acid, 800.44-11 mg/L of tween and 450mg/L of F-6818-sodium;

the concentration of the glucose in the serum-free medium is 1000-5000 mg/L;

the concentration of the HEPES in the serum-free medium is 1000-7000 mg/L;

9. Use of the additive according to any one of claims 1 to 4 or the serum-free medium according to any one of claims 5 to 8 in immune cell culture.

10. The use according to claim 9, wherein the immune cells are selected from PBMCs or T cells.