CN106520692A - Culture medium for in-vitro culture of immune cells and preparation method thereof - Google Patents
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Abstract
The invention belongs to the technical field of cell culture and, particularly, relates to a culture medium for in-vitro culture of immune cells and a preparation method thereof. The culture medium of the invention comprises a basic medium and an additive added to the basic medium; the additive is prepared from, by final concentration, 30-42 mg/L of gelatin, 8-12 Mum of D-calcium pantothenate, 30-40 mg/L of sericin, 40-60 Mum of sodium selenite, 7-15 Mug/L of fibroblast growth factor, 2-5 mg/L of glucagon, 2-4 Mug/L of reduced glutathione, 1.5-2.5 mg/L of folic acid, 0.02-0.08 mg/L of lipoic acid, 10-14 mg/L of adenosine deaminase, and 3-5 mg/L of astragaloside. The culture medium of the invention has clear composition and low price, and is capable of significantly increasing the proliferation speed of immune cells.
Description
Technical field
The invention belongs to technical field of cell culture, and in particular to a kind of culture medium for immunocyte In vitro culture and
Its preparation method.
Background technology
Research shows, immunocyte under emergency rating rapidly amplification be immunne response to environmental change done should
Answer, so as to playing a part of phagocytosis, killing and remove xenobiotic.But for cancerous issue, tumor cell can have exempts from
The characteristics of epidemic disease is escaped, you can to escape normal immune defense system, and then excessively growth forms tumor tissues in vivo.With
The rise of immunocyte personalization technology, it is now recognized that the immunity of external rapid amplifying native form immunocyte or genetic modification
Feed back to suckling after cell again biological in vivo, be conducive to the immune defence ability of raising body, show that can improve body resists
The disease treatment ability such as tumor, the ability for making histoorgan rejuvenation and prolongation organism life-span.
Chinese patent application CN105936891A discloses a kind of culture medium for immunocyte In vitro culture, including nothing
Serum basal medium, blood plasma, cytokine and Cistanchis glycosides.The content of the Cistanchis glycosides be 5-15mg/mL, the blood plasma
Volume ratio with serum-free basal medium is 1：15-1：25, the cytokine include interleukin, monoclonal antibody and
Interferon, the interleukin be interleukin-2, interleukin-4, IL-5, IL-12, interleukin-15, interleukin-
One or more in 23, the monoclonal antibody is anti-cd 3 antibodies, one or more in anti-CD28 antibody, the interference
Element is gamma interferon.
Chinese patent application CN105176925A discloses a kind of immunocyte serum-free medium and its preparation and use,
The main component of the serum-free medium of the invention is：Dexamethasone, human transferrin, insulin, cholesterol, hydrogen peroxide
Enzyme, sodium selenite and 3-mercaptoethanol.
At present, although have been developed for immunocyte serum-free medium, but there is expensive, culture immunocyte
When its growth rate it is slow the problems such as.Therefore, develop a kind of the thin for immunity of relatively low, the suitable immunocyte fast breeding of price
The culture medium of born of the same parents' In vitro culture is necessary.
The content of the invention
For the deficiencies in the prior art, it is an object of the invention to provide a kind of culture for immunocyte In vitro culture
Base and preparation method thereof.The present invention is clear and definite for the medium component of immunocyte In vitro culture and price is relatively low, can provide thin
Sufficient nutrition and good environment needed for intracellular growth propagation, can significantly improve the amplification rate of immunocyte, make immunocyte
The time of In vitro culture substantially shortens.
The technical scheme is that：
A kind of culture medium for immunocyte In vitro culture, including basal medium and being added on the basal medium
In additive, the composition of the additive with final concentration, including：Gelatin 30-42mg/L, 8-12 μM of D-VB5 calcium, sericin
Albumen 30-40mg/L, 40-60 μM of sodium selenite, fibroblast growth factor (FGF) 7-15 μ g/L, glucagon 2-5mg/L, reduction
Type glutathion 2-4 μ g/L, Folic Acid 1.5-2.5mg/L, thioctic acid 0.02-0.08mg/L, ADA Adenosine deaminase 10-14mg/L are yellow
Stilbene first glycosides 3-5mg/L.
A kind of culture medium for immunocyte In vitro culture, preferred scheme is, including basal medium and is added on
Additive in the basal medium, the composition of the additive with final concentration, including：Gelatin 35mg/L, D-VB5 calcium 9
μM, sericin 33mg/L, 53 μM of sodium selenite, 10 μ g/L of fibroblast growth factor (FGF), glucagon 4mg/L, reduced form
3 μ g/L of glutathion, Folic Acid 1.8mg/L, thioctic acid 0.05mg/L, ADA Adenosine deaminase 12mg/L, astragaloside 4mg/L.
The basal medium is DMEM or F12 culture medium.
In addition, present invention also offers the preparation method of the culture medium for immunocyte In vitro culture, step is such as
Under：
S1 adds D-VB5 calcium, sericin, sodium selenite, fibroblast growth factor (FGF), pancreas high in basal medium
Blood glucose element, reduced glutathion, Folic Acid, thioctic acid and astragaloside, stir 25-40 minutes, add ADA Adenosine deaminase, continue
Stirring 15-25 minutes, gelatin is added, stir 1 hour, obtain mixture；
The pH to 7.0-7.5 of mixture obtained by S2 regulating step S1, with 0.2-0.25 micron membrane filter filtration sterilizations, obtains final product.
Preferably, step S1 is stirred 35 minutes.
Preferably, step S1 continues stirring 22 minutes.
Preferably, the pH to 7.3 of mixture obtained by the step S2 regulating step S1.
Preferably, step S2 is with 0.21 micron membrane filter filtration sterilization.
Culture medium of the present invention for immunocyte In vitro culture, cultivates with the basis is added on including basal medium
Additive in base, the additive being added in the basal medium include gelatin, D-VB5 calcium, sericin and Radix Astragali first
Glycosides etc..In culture medium of the present invention, each composition is mutually coordinated, collective effect, there is provided the sufficient nutrition needed for growth and proliferation of cell with it is good
Good environment, can significantly improve the amplification rate of immunocyte, greatly shorten the time of immunocyte culture.
Compared with prior art, have the advantage that provided by the present invention for the culture medium of immunocyte In vitro culture：
(1) it is clear and definite provided by the present invention for the medium component of immunocyte In vitro culture and price is relatively low, can provide
Sufficient nutrition and good environment needed for growth and proliferation of cell, can significantly improve the amplification rate of immunocyte, greatly shorten
The time of immunocyte culture.
(2) culture medium provided by the present invention for immunocyte In vitro culture does not contain hyclone and without any dynamic
Thing source composition, improves clinical safety.
Specific embodiment
Below by way of the description of specific embodiment, the invention will be further described, but this is not the limit to the present invention
System, those skilled in the art's basic thought of the invention, various modifications may be made or improves, but without departing from this
The basic thought of invention, within the scope of the present invention.
Gelatin used by of the invention is purchased from the safe and sound bio tech ltd in Shenzhen, and it is public that DMEM culture medium is purchased from Sigma
Department, F12 culture medium are purchased from Sigma companies, and astragaloside is purchased from Huizhou City legendary god of farming book on Chinese herbal medicine health-care products company limited.
Embodiment 1, a kind of culture medium for immunocyte In vitro culture
The culture medium for immunocyte In vitro culture, including basal medium and being added on the basal medium
In additive, the composition of additive with final concentration, including：Gelatin 30mg/L, 8 μM of D-VB5 calcium, sericin 30mg/L,
40 μM of sodium selenite, 7 μ g/L of fibroblast growth factor (FGF), glucagon 2mg/L, 2 μ g/L of reduced glutathion, Folic Acid
1.5mg/L, thioctic acid 0.02mg/L, ADA Adenosine deaminase 10mg/L, astragaloside 3mg/L.
The basal medium is F12 culture medium.
Preparation method：
S1 adds D-VB5 calcium, sericin, sodium selenite, fibroblast growth factor (FGF), pancreas high in basal medium
Blood glucose element, reduced glutathion, Folic Acid, thioctic acid and astragaloside, stir 25 minutes, add ADA Adenosine deaminase, continue to stir
Mix 15 minutes, add gelatin, stir 1 hour, obtain mixture；
The pH to 7.0 of mixture obtained by S2 regulating step S1, with 0.2 micron membrane filter filtration sterilization, obtains final product.
Embodiment 2, a kind of culture medium for immunocyte In vitro culture
The culture medium for immunocyte In vitro culture, including basal medium and being added on the basal medium
In additive, the composition of the additive with final concentration, including：Gelatin 42mg/L, 12 μM of D-VB5 calcium, sericin
40mg/L, 60 μM of sodium selenite, 15 μ g/L of fibroblast growth factor (FGF), glucagon 5mg/L, 4 μ g/ of reduced glutathion
L, Folic Acid 2.5mg/L, thioctic acid 0.08mg/L, ADA Adenosine deaminase 14mg/L, astragaloside 5mg/L.
The basal medium is F12 culture medium.
Preparation method：
S1 adds D-VB5 calcium, sericin, sodium selenite, fibroblast growth factor (FGF), pancreas high in basal medium
Blood glucose element, reduced glutathion, Folic Acid, thioctic acid and astragaloside, stir 40 minutes, add ADA Adenosine deaminase, continue to stir
Mix 25 minutes, add gelatin, stir 1 hour, obtain mixture；
The pH to 7.5 of mixture obtained by S2 regulating step S1, with 0.25 micron membrane filter filtration sterilization, obtains final product.
Embodiment 3, a kind of culture medium for immunocyte In vitro culture
The culture medium for immunocyte In vitro culture, including basal medium and being added on the basal medium
In additive, the composition of the additive with final concentration, including：Gelatin 35mg/L, 9 μM of D-VB5 calcium, sericin
33mg/L, 53 μM of sodium selenite, 10 μ g/L of fibroblast growth factor (FGF), glucagon 4mg/L, 3 μ g/ of reduced glutathion
L, Folic Acid 1.8mg/L, thioctic acid 0.05mg/L, ADA Adenosine deaminase 12mg/L, astragaloside 4mg/L.
The basal medium is DMEM culture medium.
Preparation method：
S1 adds D-VB5 calcium, sericin, sodium selenite, fibroblast growth factor (FGF), pancreas high in basal medium
Blood glucose element, reduced glutathion, Folic Acid, thioctic acid and astragaloside, stir 35 minutes, add ADA Adenosine deaminase, continue to stir
Mix 22 minutes, add gelatin, stir 1 hour, obtain mixture；
The pH to 7.3 of mixture obtained by S2 regulating step S1, with 0.21 micron membrane filter filtration sterilization, obtains final product.
Comparative example 1, a kind of culture medium for immunocyte In vitro culture
The culture medium for immunocyte In vitro culture, including basal medium and being added on the basal medium
In additive, the composition of the additive with final concentration, including：Gelatin 35mg/L, 9 μM of D-VB5 calcium, sericin
33mg/L, 53 μM of sodium selenite, 10 μ g/L of fibroblast growth factor (FGF), glucagon 4mg/L, 3 μ g/ of reduced glutathion
L, Folic Acid 1.8mg/L, thioctic acid 0.05mg/L, ADA Adenosine deaminase 12mg/L, phillyrin 4mg/L.
The basal medium is DMEM culture medium.
Preparation method is similar to Example 3.
Difference with embodiment 3 is that astragaloside is replaced with phillyrin.
Comparative example 2, a kind of culture medium for immunocyte In vitro culture
It is described including basal medium and the additive that is added in the basal medium, the composition of the additive with
Final concentration, including：Gelatin 35mg/L, 9 μM of D-VB5 calcium, sericin 33mg/L, 53 μM of sodium selenite, fibroblastic growth
10 μ g/L of the factor, glucagon 4mg/L, 3 μ g/L of reduced glutathion, Folic Acid 1.8mg/L, thioctic acid 0.05mg/L, gland
Guanosine deaminase 12mg/L, Herba Cistanches total glycosides 4mg/L.
The basal medium is DMEM culture medium.
Preparation method is similar to Example 3.
Difference with embodiment 3 is that astragaloside is replaced with Herba Cistanches total glycosides.
Test example one, present invention gained are used for culture effect of the culture medium of immunocyte In vitro culture to immunocyte
Tested culture medium：It is used for the culture medium of immunocyte In vitro culture, and comparative example obtained by embodiment of the present invention 1-3
1 and comparative example 2 obtained by for immunocyte In vitro culture culture medium.
Test method：Cell culture experiments in vitro is done from the T cell of people source, cell presses 104It is separately added into after inoculation
Embodiment of the present invention 1-3 is used for the culture medium of immunocyte In vitro culture, and comparative example 1 and comparative example 2 are external for immunocyte
The culture medium of culture cultivated (37 DEG C, 5%CO2), after cultivating 14 days, cell quantity is determined, as a result as shown in table 1.
Table 1：Different culture media is to cell quantity after immunocyte culture
As can be seen from Table 1, in identical incubation time, it is grown in being used for immunity obtained by embodiment of the present invention 1-3 carefully
The cell quantity of immunocyte in the culture medium of born of the same parents' In vitro culture, hence it is evident that more than being grown in being used for obtained by comparative example 1 and comparative example 2
The cell quantity of immunocyte in the culture medium of immunocyte In vitro culture.This explanation, the present invention are trained in vitro for immunocyte
Foster culture medium can be obviously promoted the propagation of immunocyte, amplification of the present invention for the culture medium of immunocyte In vitro culture
Effect is significant.
Claims (8)
1. a kind of culture medium for immunocyte In vitro culture, it is characterised in that including basal medium and be added on described
Additive in basal medium, the composition of the additive with final concentration, including：Gelatin 30-42mg/L, D-VB5 calcium 8-
12 μM, sericin 30-40mg/L, 40-60 μM of sodium selenite, fibroblast growth factor (FGF) 7-15 μ g/L, glucagon 2-
5mg/L, reduced glutathion 2-4 μ g/L, Folic Acid 1.5-2.5mg/L, thioctic acid 0.02-0.08mg/L, ADA Adenosine deaminase 10-
14mg/L, astragaloside 3-5mg/L.
2. the culture medium of immunocyte In vitro culture is used for as claimed in claim 1, it is characterised in that including basal medium
With the additive being added in the basal medium, the composition of the additive with final concentration, including：Gelatin 35mg/L,
9 μM of D-VB5 calcium, sericin 33mg/L, 53 μM of sodium selenite, 10 μ g/L of fibroblast growth factor (FGF), glucagon 4mg/
L, 3 μ g/L of reduced glutathion, Folic Acid 1.8mg/L, thioctic acid 0.05mg/L, ADA Adenosine deaminase 12mg/L, astragaloside
4mg/L。
3. the preparation method of the culture medium of immunocyte In vitro culture is used for as claimed in claim 1 or 2, it is characterised in that
The basal medium is DMEM or F12 culture medium.
4. the preparation method of the culture medium for immunocyte In vitro culture as described in claim 1-3 is arbitrary, its feature exist
In step is as follows：
S1 adds D-VB5 calcium, sericin, sodium selenite, fibroblast growth factor (FGF), pancreas hyperglycemia in basal medium
Element, reduced glutathion, Folic Acid, thioctic acid and astragaloside, stir 25-40 minutes, add ADA Adenosine deaminase, continue stirring
15-25 minutes, gelatin is added, stir 1 hour, obtain mixture；
The pH to 7.0-7.5 of mixture obtained by S2 regulating step S1, with 0.2-0.25 micron membrane filter filtration sterilizations, obtains final product.
5. the preparation method of the culture medium of immunocyte In vitro culture is used for as claimed in claim 4, it is characterised in that described
Step S1 is stirred 35 minutes.
6. the preparation method of the culture medium of immunocyte In vitro culture is used for as claimed in claim 4, it is characterised in that described
Step S1 continues stirring 22 minutes.
7. the preparation method of the culture medium of immunocyte In vitro culture is used for as claimed in claim 4, it is characterised in that described
The pH to 7.3 of mixture obtained by step S2 regulating step S1.
8. the preparation method of the culture medium of immunocyte In vitro culture is used for as claimed in claim 4, it is characterised in that described
Step S2 is with 0.21 micron membrane filter filtration sterilization.
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| CN201611238566.3A CN106520692A (en) | 2016-12-28 | 2016-12-28 | Culture medium for in-vitro culture of immune cells and preparation method thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104694470A (en) * | 2015-03-31 | 2015-06-10 | 彭乐 | Serum-free medium for stem cells |
| CN104818248A (en) * | 2015-03-25 | 2015-08-05 | 苏州佰通生物科技有限公司 | Immunocyte culture medium, and culture method and application of immunocytes |
| CN105176925A (en) * | 2015-09-26 | 2015-12-23 | 友康恒业生物科技（北京）有限公司 | Immune cell serum-free medium and preparation and application thereof |
-
2016
- 2016-12-28 CN CN201611238566.3A patent/CN106520692A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104818248A (en) * | 2015-03-25 | 2015-08-05 | 苏州佰通生物科技有限公司 | Immunocyte culture medium, and culture method and application of immunocytes |
| CN104694470A (en) * | 2015-03-31 | 2015-06-10 | 彭乐 | Serum-free medium for stem cells |
| CN105176925A (en) * | 2015-09-26 | 2015-12-23 | 友康恒业生物科技（北京）有限公司 | Immune cell serum-free medium and preparation and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| XIANGFENG KONG等: "Effects of Chinese herbal medicinal ingredients on peripheral lymphocyte proliferation and serum antibody titer after vaccination in chicken", 《INTERNATIONAL IMMUNOPHARMACOLOGY》 * |
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