CN103060270A

CN103060270A - Monocyte space culture medium and preparation method thereof

Info

Publication number: CN103060270A
Application number: CN2012103796199A
Authority: CN
Inventors: 刘长庭; 郭英华; 郭娜; 方向群; 王俊峰; 李天志; 常德; 苏龙翔; 陈振鸿; 王立
Original assignee: Chinese PLA General Hospital
Current assignee: Chinese PLA General Hospital
Priority date: 2012-09-26
Filing date: 2012-09-26
Publication date: 2013-04-24
Anticipated expiration: 2032-09-26
Also published as: CN103060270B

Abstract

The invention relates to the technical field of biology, particularly preparation of a monocyte space culture medium. The preparation method comprises the following step: mixing 1640 basal culture medium free of L-glutamine and HEPES, 3g/L L-glutamine, 1.5mg/L hydrocortisone, fetal calf serum and 1000U/ml penicillin and streptomycin according to certain percentage to obtain the monocyte space culture medium. The invention ensures long-time passive growth of monocytes in a space environment; and the invention ensures the monocytes to resist low temperature, intense radiation, micro gravity and other harsh environments in the long-time space simulated environment without greatly changing the raw materials of the existing culture medium, and ensures the moderate growth and lowered apoptosis rate, thereby providing necessary conditions for research in the field of space medicine cells.

Description

A kind of monocyte space substratum and preparation method thereof

Technical field

The present invention relates to a kind of preparation of monocyte space substratum, belong to the astroplankton technical field, guarantee that monocyte resists the unfavorable factor in the space environment, appropriate growth, reduce apoptosis rate, thereby provide essential condition for the research of aeromedicine cell field.

Background technology

Space environment is complicated and changeable, is the severe environment of a low temperature, severe radiation, microgravity.The cosmonaut of operation will be in the face of complicated external environment in space environment, studies verified these environmental factorss and can exert an influence to cosmonaut's health.For further exploring its impact mechanism and formulating Counter-measures, need to be from comprehensive conducting a research such as human body, tissue, cell and experimentation on animalies.Along with the development of Chinese Space medical science, the progress of manned spaceflight technology, space environment is more and more to the research of human body aspect, and the research of cell field is also extensively carried out.No matter be ground simulation space environment or space treatment, still there are many bottleneck problems in the cell research field, especially still have many technical barriers that are difficult to capture at cell culture medium aspect preparing, for example cell is in long-term space flight, how to resist abominable space environment, utilize limited culture condition to survive, keep the balance of proliferation and apoptosis?

In the ground experiment chamber, it is ripe that the method for cell cultures has been tending towards at present, according to the difference of cytotrophy state, cell cycle, culture condition, selects appropriate time point to change fresh cell culture medium, carries out passage, preserves cell etc.Generally, such as people's mononuclear macrophage (THP-1), changed a cell culture medium in 2-3 days, according to cell density (10 ⁶-10 ⁷/ mL) go down to posterity, went down to posterity once in 4-5 days under 37 ℃, the culture condition of 5%CO2, when preserving cell, need prepare the special cells frozen storing liquid that contains dimethyl sulfoxide (DMSO), cell is kept in the liquid nitrogen.The condition that cell cultures will reach usually in the ground experiment chamber comprises: regularly replace fresh cell culture medium, keep certain cell density and have suitable Growth of Cells condition (37 ℃, 5%CO2 cell culture incubator).Yet in space treatment experiment, because the impact of the factors such as microgravity and radiation, can't reach the condition of the suitable cells survival the same with ground, so space is cultivated and is had singularity.Therefore operability, minimizing space manpower consumption, reduction Pollution risk in order to increase the space cell culture experiment just require the replacement frequency of substratum to reduce, and even do not change cell culture medium.In outer space experiment, whole cell cultivation process is a closed environment, this just requires substratum is upgraded, develop the novel culture medium that is fit to the existence of cell space, in existing research and development achievement, some space substratum has been successfully applied in the outer space experiment of the attached cells such as tumour cell, stem cell, but the research and development for monocyte space substratum lack specificity, monocyte belongs to suspension cell, its growth cycle and growth characteristics have its uniqueness, and the space substratum of having researched and developed can not reach its best growth conditions.

Summary of the invention

The object of the invention is: overcome the defective of prior art, a kind of monocyte substratum that adapts to space environment that has is provided, essential study condition can be provided for the research of aeromedicine cell field.

Among the present invention following material is mixed with monocyte space substratum by certain percentage: 35%～45% without L-glutaminate, without 1640 basic mediums of HEPES, 4%～5% 3g/LL-glutamine, 0.01%～0.015% 1.5mg/L hydrocortisone, 50%～60% foetal calf serum, 1% 1000U/ml penicillin and Streptomycin sulphate.The basic step of preparation monocyte space substratum is: to being the 1.5mg/L hydrocortisone without adding successively L-glutaminate and the concentration that concentration is 3g/L in L-glutaminate, 1640 substratum without HEPES, be formulated as serum-free monocyte space substratum; Get again foetal calf serum and penicillin and Streptomycin sulphate in the ratio of above-mentioned space substratum and join in the serum-free monocyte space substratum, fully mix; Said process is all finished at Bechtop, strictly observes the aseptic technique principle, and the substratum for preparing is put 4 ℃ and stored for future use.After preparing successfully, monocyte is inoculated in the space substratum: at first with 37 ℃ of rewarmings of substratum, secondly the centrifugal 5min of monocyte that with the speed of 800r/min～1000r/min ground is cultivated carries out cell harvesting, blood cell counting plate counting cells quantity, density according to 105/mL is inoculated in cell in the loading device that the space substratum is housed at last, airtight preservation transportation.

The present invention is by improving the ratio of foetal calf serum in the substratum, serum-concentration reaches 50%, can greatly improve the monocytic survival time, be conducive to the balance between the monocyte maintenance Proliferation and apoptosis, after the monocyte that utilizes the present invention to cultivate was survived 397 hours under space environment, the microscopically observation of cell.

Description of drawings

Fig. 1 is the compound method schema of monocyte space substratum;

Fig. 2 is under the ordinary optical microscope, and photo (* 100 times) is cultivated on normal monocyte ground;

Fig. 3 is under the ordinary optical microscope, space treatment flight monocyte photo (* 100 times) after 397 hours.

Embodiment

Be mixed with monocyte space substratum by certain percentage among the present invention: 35%～45% without L-glutaminate, without 1640 basic mediums of HEPES, 4%～5% 3g/L L-glutaminate, 0.01%～0.015% 1.5mg/L hydrocortisone, 50%～60% foetal calf serum, 1% 1000U/ml penicillin and Streptomycin sulphate.

1640 basic mediums are guaranteed the necessary nutritive substance of Growth of Cells as the basic microenvironment that monocyte grows in the space substratum; Suitably strengthened the ratio of L-glutaminate, for nucleic acid, protein in the monocyte process of growth provide more sufficient raw material; Hydrocortisone (HC) is a kind of adrenocortical hormones material, and low dose of HC (much smaller than physiological dose) not only can grow by Promote cell's growth, and the performance anti-inflammatory action; Penicillin and streptomycin has been brought into play the effect of prevention bacterial contamination; The most important thing is foetal calf serum and concentration proportioning thereof in the space substratum, serum contains abundant nutrition, the foetal calf serum quality is the highest, contained minimum to the harmful composition of cell, and the various plasma proteinss in the serum, polypeptide, fat, carbohydrate, somatomedin, hormone, inorganics etc. are coordinated the Growth of Cells balance.Usually to select serum-concentration be 10%～20% to cell cultures in the ground environment, among the present invention serum-concentration is adjusted into 50%～60%, mainly considers in passive environment, for a long time growth of cell can consume a large amount of nutritive substances, nutritive substance is abundant in the serum, therefore improve serum proportion.

For example: the basic step of preparation 500mL monocyte space substratum is:

1. buy without L-glutaminate, without 1640 cell culture mediums of HEPES from Gibco company by described, get 225mL without L-glutaminate, without 1640 substratum of HEPES in 45% ratio, the 3g/L L-glutaminate of 25mL, 0.075mL 1.5mg/L hydrocortisone (in 0.015% ratio), be formulated as serum-free cell space substratum and amount to 250.075mL;

2. get again 250mL foetal calf serum and penicillin and Streptomycin sulphate 1mL (in 1% ratio) in described 50% ratio and join in the serum-free cell space substratum, fully mix;

3. said process is all finished at Bechtop, strictly observes the aseptic technique principle, and the substratum for preparing is put 4 ℃ and stored for future use.

Before carrying monocyte space substratum is placed on rewarming in 37 ℃ of water baths, according to the loading device volume, gets the monocyte space substratum of 500 μ L and put into loading device.Get the good monocyte of growth conditions, as shown in Figure 1: cellular form size rule, become spheroidal, be a bunch arrangement, like the grape cluster pearl more.Utilize the horizontal rotor whizzer with the speed of 1000r/min with the centrifugal 5min collecting cell of monocyte, the blood cell counting plate counting cells is by 10 ⁵The cell density of individual/mL is inoculated in cell in the loading device that adds monokaryon space substratum, airtight preservation transportation.With No. eight airship space flights in Divine Land 397 hours.After space flight finishes, observation of cell under the ordinary optical microscope, as shown in Figure 2: the part cell still keeps original size, proterties is more regular, becomes spherical shape, and the cell transparence is better, the cell that part pyknosis death is also arranged in the visual field, the dead cell volume obviously diminishes than survivaling cell, and cell becomes the black lumps, without light transmission.

The non-elaborated part of the present invention belongs to techniques well known.

Claims

1. monocyte space substratum, it is characterized in that: monocyte space substratum is comprised of by certain percentage following material: 35%～45% without L-glutaminate, without 1640 basic mediums of HEPES, 4%～5% 3g/L L-glutaminate, 0.01%～0.015% 1.5mg/L hydrocortisone, 50%～60% foetal calf serum, 1% 1000U/ml penicillin and Streptomycin sulphate.

2. prepare 500mL monocyte space claimed in claim 1 substratum, it is characterized in that: required above-mentioned substance is respectively: 225mL without L-glutaminate, without 1640 basic mediums of HEPES, the 3g/L L-glutaminate of 25mL, 0.075mL the 1.5mg/L hydrocortisone, the foetal calf serum of 250mL, 1000U/mL penicillin and the Streptomycin sulphate of 1mL.

3. the preparation method of monocyte space substratum, it is characterized in that: the specific configuration process is:

1. to being the 1.5mg/L hydrocortisone without adding successively L-glutaminate and the concentration that concentration is 3g/L in L-glutaminate, 1640 substratum without HEPES, be formulated as serum-free monocyte space substratum;

2. get again foetal calf serum and penicillin and Streptomycin sulphate in ratio claimed in claim 1 and join in the serum-free monocyte space substratum, fully mix;

4. utilize claim 1 and 2 described monocyte space substratum, it is characterized in that: cultivating monocytic concrete steps is: at first with 37 ℃ of rewarmings of substratum, secondly the centrifugal 5min of monocyte that with the speed of 800r/min～1000r/min ground is cultivated carries out cell harvesting, blood cell counting plate counting cells quantity is at last according to 10 ⁵The density of individual/mL is inoculated in cell in the loading device that the space substratum is housed, airtight preservation transportation.