CN108029678A - Cell preservation method and cell transportation resources - Google Patents
Cell preservation method and cell transportation resources Download PDFInfo
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- CN108029678A CN108029678A CN201711465084.6A CN201711465084A CN108029678A CN 108029678 A CN108029678 A CN 108029678A CN 201711465084 A CN201711465084 A CN 201711465084A CN 108029678 A CN108029678 A CN 108029678A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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Abstract
The present invention relates to biological technical field, provide a kind of cell preservation method for preserving living cells and cell transportation resources, in particular it relates to a kind of combination cell in the state of cell function is maintained preserves cell preservation method and the cell transportation resources that liquid preserves living cells.The mode of cell preservation method of the present invention is to preserve liquid using the cell culture apparatus with multiple micro- spaces and combination cell to protect the cell preservation method of living cells, living cells is set to be adhered to the surface in above-mentioned multiple micro- spaces to be cultivated, after above-mentioned culture, 8%~20%HES (hydroxyethyl starch) will be contained, 50mg/L~150mg/L streptomysins, the culture medium of 10%~30%DMSO (dimethyl sulfoxide (DMSO)) cell-preservation liquid is injected to above-mentioned cell culture apparatus, to cover above-mentioned multiple micro- spaces, above-mentioned cell culture container is sealed, so that culture medium does not leak out, preserved, can with maintain the state of function preserve living cells.
Description
Technical field
The invention belongs to biological technical field, is related to a kind of cell preservation method and cell transporter for preserving living cells
Method.
Background technology
In the preservation of blood, rare cells and bio-tissue etc., the traditional cooling Cord blood at a slow speed of generally use
Method, i.e. " two-step method " are preserved.The particularly rare cells such as bone marrow cell, candidate stem cell, mescenchymal stem cell, these are thin
Born of the same parents have suitable beneficial effect, it is necessary to the technology preserved for a long time to it in clinical treatment.Under this low temperature
In preservation, usually the loadings such as above-mentioned blood, rare cells and bio-tissue are received in the container for filling these materials, Ran Houmi
It is enclosed in liquid nitrogen and preserves.But influenced by cooldown rate, the cell or tissue of preservation can be caused to damage.If rate of temperature fall mistake
It hurry up, then form intracellular ice, recrystallization can occur during rewarming for these intracellular ices makes cell damage；If rate of temperature fall is excessively slow,
Then cellular contraction is excessively acute, and cell is in highly concentrated solution can also cause damage.
In histiocytic culture, due to the use of the cell culture strain for cell culture test, it is desirable to its show
With experiment in vivo, i.e., so-called (in vivo) in vivo tests same drug-susceptibility, toxic reaction, it is necessary to thin
The iuntercellular network that the surface of born of the same parents' culture vessel arranges in which can construct regular property.Further, since in cell culture test
The cell culture strain used is prohibitively expensive, it is therefore desirable for improving the survival rate and growth rate of cell.
In order to solve above-mentioned two problems, the present invention provides one kind, combination cell is protected in the state of cell function is maintained
Liquid storage preserves cell preservation method and the cell transportation resources of living cells.
The content of the invention
It is an object of the invention to provide one kind, combination cell preservation liquid preservation is living thin in the state of cell function is maintained
The cell preservation method of born of the same parents and cell transportation resources, can solve the problems, such as that existing cell-preservation liquid is influenced by cooldown rate, together
When can also solve the problems, such as it is expected to improve cells survival rate and growth rate in cell culture test.
One aspect of the present invention provides a kind of cell culture apparatus of the use with multiple micro- spaces and combination cell is protected
Liquid storage protects the cell preservation method of living cells, living cells is adhered to the surface in above-mentioned multiple micro- spaces to be cultivated,
After above-mentioned culture, 8%~20%HES (hydroxyethyl starch), 50mg/L~150mg/L streptomysins, 10%~30% will be contained
The culture medium of DMSO (dimethyl sulfoxide (DMSO)) cell-preservation liquid is injected to above-mentioned cell culture apparatus, to cover above-mentioned multiple micro- spaces,
Above-mentioned cell culture container is sealed, so that culture medium does not leak out, is preserved.Surface adhesion living cells in micro- space, makes
Cultivated with suitable for micro- space of culture, three-dimensional structure is formed in each micro- container, utilize micro- vessel isolation three-dimensional structure
After body, and combination cell preserve liquid sealed and preserved, more can with maintain the state of function preserve living cells.
In another preference, the cell culture container is placed in 4 DEG C~37 DEG C temperature conditionss, and to get off to preserve above-mentioned work thin
Born of the same parents.Preferably, the cultivation temperature is 10 DEG C~20 DEG C.
In another preference, the concentration of HES is 8%~10% in the cell-preservation liquid；In specific embodiment
In, the concentration of the HES in the culture medium is 10%.
In another preference, the concentration of the cell-preservation liquid streptomycin is 50mg/L~100mg/L；Specific
In embodiment, the concentration of the streptomysin is 80mg/L.
In another preference, the concentration of DMSO is 10%~20% in the cell-preservation liquid；In specific embodiment party
In formula, the concentration of the DMSO is 10%.
In another preference, the multiple micro- preferred bottom area in space is 0.02~0.15mm2, depth for 20~
100μm。
In another preference, the living cells is selected from the one or more of following cells：Liver cell, pancreatic β cell, the heart
Myocyte, nerve cell, skin epithelial cell, cartilage cell, osteocyte, tissue stem cell, ES cells and iPS cells；It is preferred that
Ground, the one or more in tissue stem cell, cartilage cell and osteocyte.
It is with 1~1 × 10 in another preference6Cell/cm2Cell-seeding-density by seeded with living celis to above-mentioned more
In a micro- space, it is preferable that cell-seeding-density is 1 × 104~1 × 106。
Another aspect of the present invention, there is provided a kind of cell transportation resources, be by living cells be stored in it is described have it is multiple
In the cell container in micro- space and transport method.The transportation resources makes living cells be adhered to the table in above-mentioned multiple micro- spaces first
Face is cultivated, and after above-mentioned culture, will contain 8%~20%HES (hydroxyethyl starch), 50mg/L~150mg/L strepto-s
Element, the culture medium of 10%~30%DMSO (dimethyl sulfoxide (DMSO)) cell-preservation liquid are injected to above-mentioned cell culture apparatus, with covering
Multiple micro- spaces are stated, above-mentioned cell culture container is sealed, so that culture medium does not leak out.It is optional, using vehicle, ship or
Any of aircraft conveying arrangement is transported the above-mentioned cell culture container of sealing.
In another preference, the concentration of HES is 8%~10% in the cell-preservation liquid；In specific embodiment
In, the concentration of the HES is 10%.
In another preference, the concentration of the cell-preservation liquid streptomycin is 50mg/L~100mg/L；Specific
In embodiment, the concentration of the streptomysin is 80mg/L.
In another preference, the concentration of DMSO is 10%~20% in the cell-preservation liquid；In specific embodiment party
In formula, the concentration of the DMSO is 10%.
According to the present invention it is possible to providing the cell culture apparatus with multiple micro- spaces and combination cell preserves liquid to protect work
The cell preservation method of cell and cell transportation resources.
Advantageous effect of the invention
It is of the present invention to use the cell culture apparatus with multiple micro- spaces, and combination cell preserves liquid and lives carefully to protect
The cell preservation method of born of the same parents, because it combines the structure in micro- space and protective effect of the cell-preservation liquid to cytoactive, more can
The stability and cytoactive of living cells are enough kept, cell is effectively applied.
Brief description of the drawings
Fig. 1 show influence of the different store methods to Cell viability.
Fig. 2 show influence of the different cell-preservation liquid formulas to Cell viability.
Embodiment
Embodiment of the present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings, but art technology
Personnel will be understood that the following example is merely to illustrate the present invention, and should not be construed as the limitation of the present invention.In following embodiments
Experimental method, unless otherwise specified, be according to normal condition or manufacturer suggestion condition carry out.In following embodiments
Material used, reagent etc., unless otherwise specified, be commercially or acquisition purchased in market conventional products.
The cell preservation method of the present invention is the cell culture container used suitable for living cells culture, is adhered to living cells
Micro- space is cultivated, and three-dimensional structure is formed in micro- space, after above-mentioned culture, fills the culture medium containing cell-preservation liquid
Preserved.The cell-preservation liquid contain 8%~20%HES (hydroxyethyl starch), 50mg/L~150mg/L streptomysins,
10%~30%DMSO (dimethyl sulfoxide (DMSO)).
Concavo-convex general layout, i.e., multiple micro- containers are formed in the cell culture container.Preferably, can be by the way that micro- container will be made
The height of the side wall (convex portion) of separation optimizes, and can only cultivate the living cells of aggregation in micro- container.Wherein, micro- space is
The space formed by micro- container, more specifically, referring to by the concavo-convex general layout formed in the plane the space that is formed.
Micro- container as described herein and micro- space are not distinguished especially.
1 cell-preservation liquid of the present invention of embodiment
(1) it is formulated
The cell-preservation liquid by 8%~20%HES (hydroxyethyl starch), 50mg/L~150mg/L streptomysins, 10%~
30%DMSO (dimethyl sulfoxide (DMSO)) is formed.
(2) preparation method
In ratio in formula, streptomysin is dissolved and is uniformly mixed with HES, DMSO is added, is placed in ice-water bath extremely
Preserve nearly 0 DEG C of liquid temperature degree.
2 cell of embodiment preserves viability experiment
Packet as shown in table 1 carries out cell and preserves viability experiment.
Table 1 is grouped
| A groups | Cell-preservation liquid |
| B groups | Cell container with multiple micro- spaces |
| C groups | Cell container+cell-preservation liquid with multiple micro- spaces |
A groups store method is that living cells is preserved only with cell-preservation liquid；B groups are only with multiple micro- skies
Between cell container living cells preserved；C groups are to be preserved using the cell container combination cell with multiple micro- spaces
Liquid preserves living cells.
3 groups of store methods preserve the cell of same source, and the cell derived is in people's fat mesenchymal
Stem cell, in 37 DEG C, 5%CO2Incubator internal breeding, until predetermined cell density.
The component of the cell-preservation liquid is 10%HES (hydroxyethyl starch), 80mg/L streptomysins, 10%DMSO (diformazans
Base sulfoxide).
The culture medium uses DMEM culture mediums, is peeled off the cell of propagation from culture bottom surface using 0.25% pancreatin, with
Centrifugal separation recycles cell.
Using above-mentioned 3 kinds of store methods, preserved 3 days under 10 DEG C of temperature conditionss.The cell after preserving 3 days is calculated respectively to live
Rate.The results are shown in Figure 1, C group Cell viability highests.It follows that using the cell culture apparatus with multiple micro- spaces, and tie
Cell-preservation liquid is closed to protect living cells to be best able to maintain the stability and cytoactive of living cells.
The packet of 3 different formulations of embodiment carries out cell and preserves viability experiment
The different cell-preservation liquid formula as shown in table 2 is combined with the cell culture apparatus with multiple micro- spaces carries out cell
Preserve viability experiment.
2 cell-preservation liquid formula of table
| A groups | 8%HES, 50mg/L streptomysin, 30%DMSO |
| B groups | 10%HES, 100mg/L streptomysin, 20%DMSO |
| C groups | 20%HES, 150mg/L streptomysin, 10%DMSO |
In addition to formula of liquid difference is preserved, other conditions all same.
After being preserved 3 days under 10 DEG C of temperature conditionss, the Cell viability of 3 groups is calculated respectively.The results are shown in Figure 2, thus may be used
Know, the culture medium containing 10%HES, 100mg/L streptomysin, the cell-preservation liquid of 20%DMSO, and with the thin of multiple micro- spaces
Born of the same parents' culture vessel combines, and there is highest cell to preserve activity and stability.It follows that the use of the present invention is with multiple micro-
The cell culture apparatus in space, and combination cell preserves liquid to protect the cell preservation method of living cells that there is higher cell to preserve
Activity, can ensure the vigor and stability of cell, cell is effectively applied.
Although the embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that root
According to disclosed all teachings, various modifications and replacement can be carried out to these embodiments, these change the present invention's
Within protection domain.The four corner of the present invention is provided by appended claims and its any equivalent.
Claims (10)
1. a kind of cell preservation method, it is characterised in that protected using the cell culture apparatus with multiple micro- spaces and combination cell
Liquid storage protects the cell preservation method of living cells.
2. living cells according to claim 1 is selected from the one or more of following cells：Liver cell, pancreatic β cell, cardiac muscle
Cell, nerve cell, skin epithelial cell, cartilage cell, osteocyte, tissue stem cell, ES cells and iPS cells；Preferably,
One or more in tissue stem cell, cartilage cell and osteocyte.
3. cell preservation method according to claim 1, it is characterised in that make living cells be adhered to above-mentioned multiple micro- spaces
Surface cultivated, after above-mentioned culture, the culture medium containing cell-preservation liquid is injected to above-mentioned cell culture apparatus, with
Above-mentioned multiple micro- spaces are covered, above-mentioned cell culture container is sealed, so that culture medium does not leak out, is preserved.
4. cell preservation method according to claim 3, it is characterised in that the cell-preservation liquid is by 8%~20%HES
(hydroxyethyl starch), 50mg/L~150mg/L streptomysins, 10%~30%DMSO (dimethyl sulfoxide (DMSO)) compositions.
5. cell preservation method according to claim 4, it is characterised in that the concentration of HES is in the cell-preservation liquid
8%~10%；Preferably, the concentration of the HES is 10%.
6. cell preservation method according to claim 4, it is characterised in that the concentration of the cell-preservation liquid streptomycin
For 50mg/L~100mg/L；Preferably, the concentration of the streptomysin is 80mg/L.
7. cell preservation method according to claim 4, it is characterised in that the concentration of DMSO is in the cell-preservation liquid
10%~20%；Preferably, the concentration of the DMSO is 10%.
8. according to claim 1-7 any one of them cell preservation methods, it is characterised in that the cell culture container is placed in
4 DEG C~37 DEG C temperature conditionss get off to preserve above-mentioned living cells；Preferably, the cultivation temperature is 10 DEG C~20 DEG C.
9. a kind of cell transportation resources, it is characterised in that living cells is stored in the cell container with multiple micro- spaces
In and transport method.
10. cell transportation resources according to claim 9, it is characterised in that be first adhered to living cells above-mentioned multiple
The surface in micro- space is cultivated, after above-mentioned culture, by the culture medium containing cell-preservation liquid to above-mentioned cell culture apparatus
Injection, to cover above-mentioned multiple micro- spaces, above-mentioned cell culture container is sealed, so that culture medium does not leak out；Preferably, it is described
Cell-preservation liquid is by 8%~20%HES (hydroxyethyl starch), 50mg/L~150mg/L streptomysins, 10%~30%DMSO (two
Methyl sulfoxide) composition.
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| CN201711465084.6A CN108029678A (en) | 2017-12-28 | 2017-12-28 | Cell preservation method and cell transportation resources |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109938010A (en) * | 2019-03-20 | 2019-06-28 | 江苏瑞思坦生物科技有限公司 | A kind of human adipose mesenchymal stem cells transport liquid and preparation method thereof |
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Application publication date: 20180515 |