CN103461322B - The preservation liquid of the cell after cryopreservation resuscitation - Google Patents
The preservation liquid of the cell after cryopreservation resuscitation Download PDFInfo
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- 238000004321 preservation Methods 0.000 title claims abstract description 40
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims abstract description 20
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 19
- 239000000203 mixture Substances 0.000 claims abstract description 16
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Abstract
The invention provides the preservation liquid of a kind of cell after cryopreservation resuscitation, including following components: normal saline, KCl and MgSO4.The invention also discloses the purposes preserving liquid, and comprise the umbilical cord stem cells mixture of this preservation liquid and umbilical cord stem cells.Using preserving liquid and can preserving the cell for clinical reinfusion after cryopreservation resuscitation for a long time of the present invention, the more traditional normal saline of cell viability preserves wants height, more meets clinical reinfusion requirement.
Description
Technical field
The present invention relates to biological technical field, in particular it relates to the preservation liquid of a kind of cell for clinical reinfusion after cryopreservation resuscitation.
Background technology
Cell therapy, treats also known as living cells, including the tissue of living cells reparation damage.Cell for clinical reinfusion is many stem cell or immunocyte.Umbilical cord contains a large amount of epithelial stem cell and mesenchymal cell.Two kinds of stem cell are respectively provided with tremendous potential and help tissue or organ, the therefore various disease of co-treatment of repair deficiency.Epithelial stem cell forms connection, supports or cover some structure of health and the soft tissue of organ, and these structures and organ include cornea, skin and liver；Interstital stem cell is then the element of structural organization (such as skeleton, cartilage, muscle, fibrous tissue and fat etc.).
Mescenchymal stem cell is derived from growing in early days the most mesoblastic has height self-renewal capacity and the pluripotent stem cell of multi-lineage potential, it is widely present in whole body Various Tissues, amplification can be cultivated in vitro, and neurocyte, osteoblast, muscle cell, adipose cell etc. can be divided under specified conditions control, in terms of cell replacement therapy and organizational project, there is great using value.At present, mescenchymal stem cell MSC (Mesenchymalstemcells, MSC) it is mainly derived from bone marrow, but owing to bone marrow derived MSC exists the possibility that highly virus is polluted, and its cell quantity and amplification, differentiation capability occur being decreased obviously trend with age, therefore find a kind of source for mesenchymal stem cells that can substitute bone marrow and its defect can be made up, the most increasingly paid close attention to by scholars.Umbilical cord mesenchymal stem cells, as a kind of pluripotent stem cell, successfully avoids many restrictions such as derived from embryonic stem cells shortage, allosome repulsion, moral ethics, becomes the study hotspot of stem cell field.
Umbilical cord MSC can separate the most the most in vitro, and amplification is rapidly, and owing to it has reduced immunogenicity, hematopoiesis support and multi-lineage potential, it has potential applicability in clinical practice widely in terms of multinomial medical tissue engineering.
Human umbilical cord mesenchymal stem cells is the preferable seed cell of organizational project.Because it draws materials conveniently except having, source is wide, abundant amount, and multiplication capacity is strong, and Long Term Passages does not changes, and secretion has the features such as the extracellular matrix that is suitably constructed.Additionally have the advantage that 1, gather umbilical cord puerpera and neonate are not had any harm and damage；2, ancestral cells is more original, has higher proliferation and differentiation ability；3, immunocyte is the inmatureest, and functional activity is low, and immunologic function is not mature enough, is in one and keeps silent state, will not trigger immunoreation and cause graft versus host disease；4, not having tumor cell, virus and the infection of pathogenic microorganism and propagation probability are relatively low；5, society, ethics and legal more dispute it are not related to.
Human umbilical cord mesenchymal stem cells not only has multi-lineage potential, it is also easy to transfection and the expression of exogenous gene, and the exogenous gene expression entrained by MSC has obvious tissue specificity, because this person umbilical cord MSCs is likely to become the target cell of a kind of novel gene therapy.By transgene method, the growth factor gene of beneficially directed differentiation can be proceeded to mescenchymal stem cell, promote directed differentiation, accelerate the process that in-vivo tissue is repaired.
But, the cell after cryopreservation resuscitation, cell membrane is the most fragile, if now selecting conventional clinical reinfusion reagent physiological saline to preserve liquid as the feedback of cell, cell is when transporting for a long time, and cell is easily downright bad and makes activity reduction.Therefore, still need and the preservation liquid of a kind of cell is provided, keep in transit its biological activity with the cell after ensureing cryopreservation resuscitation, can preferably obtain highly active cell.
Summary of the invention
It is an object of the invention to overcome the defect of prior art, it is provided that cell-preservation liquid after a kind of novel cryopreservation resuscitation and application thereof, and comprise the umbilical cord stem cells mixture of this preservation liquid and umbilical cord stem cells.
A first aspect of the present invention, it is provided that a kind of cell-preservation liquid, including following components: normal saline, KCl and MgSO4。
In another preference, the concentration of described KCl is 0.3-0.4g/l, MgSO4Concentration be 0.02-0.1g/l.
In another preference, the concentration of described KCl is 0.32-0.36g/l, MgSO4Concentration be 0.04-0.06g/l.
A second aspect of the present invention, it is provided that the purposes of the cell-preservation liquid described in first aspect, described purposes is selected from lower group:
A () is used for preserving stem cell；
B () is used for setting up stem cell bank；
C () is for keeping the vigor of stem cell
In another preference, described stem cell is the cell after cryopreservation resuscitation.
In another preference, described stem cell is umbilical cord stem cells.
In another preference, described stem cell is the umbilical cord stem cells after cryopreservation resuscitation.
In another preference, described preservation refers to preserve at 2 ~ 6 DEG C.
In another preference, described preservation refers to preserve at 3.5-4.5 DEG C.
A third aspect of the present invention, it is provided that a kind of umbilical cord stem cells mixture, described mixture includes following components:
Umbilical cord stem cells, and
Cell-preservation liquid described in first aspect.
A fourth aspect of the present invention, it is provided that a kind of umbilical cord stem cells storehouse, it is characterised in that described cell bank contains the umbilical cord stem cells mixture described in the third aspect.
Using preserving liquid and can preserving the cell after cryopreservation resuscitation for a long time of the present invention, the more traditional normal saline of cell viability preserves wants height, more meets clinical reinfusion requirement.
In should be understood that within the scope of the present invention, can be combined with each other between above-mentioned each technical characteristic and each technical characteristic specifically described in below (eg embodiment) of the present invention, thus constitute new or preferred technical scheme.As space is limited, the most tired at this state.
Accompanying drawing explanation
Fig. 1 is experiment a group and compares a group cellular morphology figure at different incubation times.
Detailed description of the invention
Present inventor, through extensively and in depth studying, finds first, uses and comprises normal saline, KCl and MgSO4Preservation liquid preserve cell, such as umbilical cord stem cells, after long-time preservation, umbilical cord stem cells is difficult to necrosis, still has good activity and reproductive performance.On this basis, the present invention is completed.
Cell-preservation liquid
The cell-preservation liquid of the present invention, including following components: normal saline, KCl and MgSO4。
In another preference, the concentration of described KCl is 0.3-0.4g/l, MgSO4Concentration be 0.02-0.1g/l.
In another preference, the concentration of described KCl is 0.32-0.36g/l, MgSO4Concentration be 0.04-0.06g/l.
Potassium ion and sodium ion one work, and can maintain the balance of internal water and normal (wherein, potassium works, and sodium only works in extracellular) of the rhythm of the heart intracellular；The function of N&M can be damaged when potassium and sodium balance imbalance.Potassium participates in many kinds of substance metabolism, affects the activity of desmoenzyme, the osmotic pressure of the regulation inside and outside liquid of cell and acid-base balance.Magnesium is cation important in living organism, is regulation extracellular Ca2＋, and potassium, the important ion of sodium balance, is the activator of multiple enzyme.In the metabolism of calcium, vitamin C, phosphorus, sodium, potassium etc., magnesium is necessary material, plays important role during nervimuscular function normal operation, blood glucose conversion etc..
Inventors be surprised to learn that, with the addition of potassium ion in normal saline, magnesium ion makes cell-preservation liquid, can preserve cell for a long time, such as the umbilical cord stem cells after cryopreservation resuscitation, can maintain and/or improve the activity of umbilical cord stem cells, it is adaptable to the preservation transport of cell.
The stem cell preserving fluid of the present invention, may be used for:
A () preserves stem cell；
B () sets up cell bank；Or
C () keeps the vigor of stem cell.
In another preference, described stem cell is the cell after cryopreservation resuscitation.
In another preference, described stem cell is umbilical cord stem cells.
In another preference, described stem cell is the umbilical cord stem cells after cryopreservation resuscitation.
In another preference, described preservation refers to preserve at 2 ~ 6 DEG C, it is preferred that described preservation refers to preserve at 3.5-4.5 DEG C.
In the present invention, term " normal saline ", refer to Physiology Experiment or the osmotic pressure conventional clinically sodium chloride solution equal with the osmotic pressure of animal or human plasma, concentration is 0.85 ~ 0.9w/v%.
The separation of the umbilical cord stem cells of the present invention, cultivation, the frozen method that this area all can be used conventional.
Umbilical cord stem cells mixture
The umbilical cord stem cells mixture of the present invention, comprises following components:
(a) umbilical cord stem cells；And
B () cell-preservation liquid, including following components: normal saline, KCl and MgSO4。
In another preference, the concentration of described KCl is 0.3-0.4g/l, MgSO4Concentration be 0.02-0.1g/l.
In another preference, the concentration of described KCl is 0.32-0.36g/l, MgSO4Concentration be 0.04-0.06g/l.
The cell-preservation liquid major advantage of the present invention includes:
(1) liquid composition is preserved clear, without the composition harmful to cell；
(2) umbilical cord stem cells can be preserved for a long time, keep the activity of stem cell；
(3) umbilical cord stem cells survival rate is high, and multiplication capacity is strong.
The features described above that the present invention mentions, or the feature that embodiment is mentioned can be in any combination.All features disclosed in this case description can be with any composition forms use, each feature disclosed in description, can identical by any offer, impartial or similar purpose alternative characteristics replace.Therefore except there being special instruction, disclosed feature is only the impartial or general example of similar features.
Unless otherwise defined, the same meaning that all specialties used in literary composition are familiar with one skilled in the art with scientific words.Additionally, any method similar or impartial to described content and material all can be applicable in the inventive method.Preferable implementation described in literary composition only presents a demonstration with material and is used.
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are merely to illustrate the present invention rather than limit the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition, such as Sambrook et al., molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage ratio and number are percentage by weight and parts by weight.
Embodiment 1
The preparation of cell-preservation liquid
By KCl, MgSO4It is dissolved in normal saline, makes the final concentration of 0.37g/l, MgSO of KCl4Final concentration of 0.05g/l.
Embodiment 2
The preservation of human umbilical cord mesenchymal stem cells
Material agents:
1, pipet, centrifuge tube, cryopreservation tube, transfering bag, human umbilical cord mesenchymal stem cells (HUC-MSC), vaccine box
2, DMEM culture medium, normal saline, stem cell preserving fluid (embodiment 1 is prepared)
Experiment packet:
Comparison a group: 30ml normal saline+1.2 × 107Individual human umbilical cord mesenchymal stem cells
Comparison b group: 1ml normal saline+0.5 × 107Individual human umbilical cord mesenchymal stem cells
Experiment a group: 30ml preserves liquid+1.2 × 107Individual human umbilical cord mesenchymal stem cells
Experiment b group: 1ml preserves liquid+0.5 × 107Individual human umbilical cord mesenchymal stem cells
Experimental technique:
Take out the frozen human umbilical cord mesenchymal stem cells of same batch (HUCMSC), add in the DMEM culture medium preheated the most in advance, fully mix, and be divided into two parts.
Every part of cell washes twice with normal saline, preservation liquid respectively, and after washing, according to experiment packet condition, preparation compares a group, comparison b group, tests a group and experiment b group.Wherein, comparison a group and the cell of experiment a group and preservation liquid are put in 50ml transfering bag, respectively fill three bags, seal with heat-sealing machine, and comparison b group and the cell of experiment b group and preservation liquid are put in 1.5ml cryopreservation tube, respectively fill three pipes.
Transfering bag and cryopreservation tube are put into and has been loaded with in the vaccine box of ice bag preserving, and keep in vaccine box temperature at 2～6 DEG C.
Taking out counting respectively after 6h, 8h, 10h, 24h and calculate motility rate, relative increase rate, computing formula is as follows:
Cell number/initial cell number × 100% after Cell viability=preservation
Increase rate=(experimental group Cell viability-cellular control unit motility rate)/cellular control unit motility rate × 100% relatively
Table 1 human umbilical cord mesenchymal stem cells preserves the Cell viability under liquid preserves at two kinds
Result is as shown in table 1.
Additionally, the cell after preserving 6h, 8h, 10h, 24h is seeded in culture bottle cultivation 24h, microexamination pattern, result is as shown in Figure 1.
Test result indicate that of table 1 and Fig. 1, under identical storage temperature, change over time, it is higher than normal saline with the Cell viability preserving liquid washing and preservation.What the present invention used preserve liquid preserves for the transport feeding back cell, and the more traditional normal saline of cell viability preserves wants height, more meets clinical reinfusion requirement, and can transport for a long time.
The above embodiment of the present invention, as a example by umbilical cord stem cells, illustrates that the preservation liquid of the present invention can preserve umbilical cord stem cells for a long time, maintains and/or improve the activity of umbilical cord stem cells, it is adaptable to the preservation transport of cell.Additionally, the preservation liquid of the present invention can also preserve other kinds of cell for a long time, such as the mescenchymal stem cell such as fat stem cell, cord stem cell, it is possible to maintain or improve the activity of cell, it is adaptable to preserve transportation.
The all documents mentioned in the present invention are incorporated as reference the most in this application, are individually recited as with reference to like that just as each document.In addition, it is to be understood that after the above-mentioned teachings having read the present invention, the present invention can be made various changes or modifications by those skilled in the art, these equivalent form of values fall within the application appended claims limited range equally.
Claims (7)
1. a cell-preservation liquid, it is characterised in that described cell-preservation liquid is composed of the following components: normal saline, KCl and MgSO4；
Wherein, the concentration of described KCl is 0.3-0.4g/l, MgSO4Concentration be 0.02-0.1g/l.
2. cell-preservation liquid as claimed in claim 1, it is characterised in that the concentration of described KCl is 0.32-0.36g/l, MgSO4Concentration be 0.04-0.06g/l.
3. the purposes of the cell-preservation liquid described in claim 1, it is characterised in that described purposes is selected from lower group:
A () is used for preserving cell；
B () is used for setting up stem cell bank；
C () is for keeping the motility rate of cell；
Wherein, described cell is the cell after cryopreservation resuscitation, and described cell and stem cell are umbilical cord stem cells.
4. purposes as claimed in claim 3, it is characterised in that described preservation refers to preserve at 2～6 DEG C.
5. purposes as claimed in claim 3, it is characterised in that described preservation refers to preserve at 3.5-4.5 DEG C.
6. a umbilical cord stem cells mixture, it is characterised in that described mixture includes following components:
Umbilical cord stem cells, and
Cell-preservation liquid described in claim 1.
7. a umbilical cord stem cells storehouse, it is characterised in that described cell bank contains the umbilical cord stem cells mixture described in claim 6.
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| CN108925548A (en) * | 2017-05-24 | 2018-12-04 | 西比曼生物科技（香港）有限公司 | A kind of freeze-stored cell preparation and cell recovery mode |
| CN109845725A (en) * | 2019-01-25 | 2019-06-07 | 北京益华生物科技有限公司 | Cell transport saves liquid and its application |
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| WO2006057876A2 (en) * | 2004-11-22 | 2006-06-01 | Cedars-Sinai Medical Center | Methods and solutions for tissue preservation |
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