CN108575986A - A kind of preservation liquid composition and its application - Google Patents
A kind of preservation liquid composition and its application Download PDFInfo
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- CN108575986A CN108575986A CN201810379498.5A CN201810379498A CN108575986A CN 108575986 A CN108575986 A CN 108575986A CN 201810379498 A CN201810379498 A CN 201810379498A CN 108575986 A CN108575986 A CN 108575986A
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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
Abstract
The present invention provides a kind of preservation liquid composition and its application, the preservation liquid includes following component in parts by weight：80 95 parts by weight of 5 15 parts by weight of physiological saline, 36 parts by weight of human serum albumin and Multiple electrolytes injection, the preservation liquid energy for preserving the preparation of liquid composition reaches long-time room temperature and preserves umbilical cord mesenchymal stem cells vigor, and cell viability is still 80% or more after preserving for 24 hours.
Description
Technical field
The present invention relates to biomedical sectors, and in particular to a kind of preservation liquid composition and its application, more particularly to it is a kind of
Liquid composition is preserved, the umbilical cord mesenchymal stem cells of composition preserve liquid and its preserve the preservation side of umbilical cord mesenchymal stem cells
Method.
Background technology
Umbilical cord mesenchymal stem cells (MSCs) have higher differentiation potential, can be broken up to multiple directions.It bone,
There is wide potential applicability in clinical practice in terms of the organizational projects such as cartilage, muscle, tendon, ligament, nerve, liver, endothelium and cardiac muscle.Have
Report isolates MSCs from people's umbilical cord, and cell content, proliferative capacity are better than bone marrow MSCs, and immunogenicity compares bone marrow MSCs
It is low, and have many advantages, such as convenient material drawing, no ethics dispute, therefore increasingly by the concern of research workers.
MSCs, cannot be by the stem cell direct infusion of fresh separated in human body in clinical transplantation treatment, it needs elder generation
It is stored in certain time in the preservation liquid (such as 0.9% sodium chloride injection) that can be transfused, then is transfused in internal.MSCs is in vitro
In the case of vigor can decline gradually, this directly affects the stem cell population and quality of separation, how to maintain the work of in vitro MSCs
Property becomes the task of top priority.Current clinically used preservation liquid has 0.9% sodium chloride injection, 5% glucose injection, and 1%
Human serum albumin liquid etc., but MSCs activity in these preservation liquid can decline, and influence clinical therapeutic effect.
There are the following problems for current umbilical cord preserving fluid：1. preserving being made of the culture medium on basis for liquid, nutritional ingredient is multiple
It is miscellaneous, and the ingredient not necessarily be suitble to umbilical cord under ex vivo needed for；2. configuration process is cumbersome；3. the preservation effect of couple MSCs
It is bad.Find that a kind of ingredient is simple at present, the method that can effectively preserve MSCs is a kind of urgent problem to be solved.
Invention content
In order to solve the above technical problems, the present invention provides a kind of preservation liquid composition and its application, the preservation liquid group
It closes preservation liquid energy prepared by object and reaches long-time room temperature preservation umbilical cord mesenchymal stem cells, stem cell vigor, preservation is kept to do afterwards for 24 hours
Cell viability is still 80% or more.
For this purpose, present invention employs following technical schemes：
In a first aspect, the present invention provides a kind of preservation liquid composition, the preservation liquid includes such as the following group in parts by weight
Point：
Physiological saline 5-15 parts by weight
Human serum albumin 3-6 parts by weight
Multiple electrolytes injection 80-95 parts by weight.
In the present invention, pass through the combination of specific physiological saline and human serum albumin so that preserving liquid composition can be
Mescenchymal stem cell is preserved under room temperature, and rear stem cell vigor remains to, 80% or more, not generate Stem cell surface marker for 24 hours
Too much influence.
The parts by weight of the physiological saline be 5-15 parts by weight, such as can be 5 parts by weight, 6 parts by weight, 7 parts by weight,
8 parts by weight, 9 parts by weight, 10 parts by weight, 11 parts by weight, 12 parts by weight, 13 parts by weight, 14 parts by weight or 15 parts by weight.
The parts by weight of the human serum albumin are 3-6 parts, such as can be 3 parts by weight, 4 parts by weight, 5 parts by weight or 6
Parts by weight.
The parts by weight of the Multiple electrolytes injection are 80-95 parts by weight, such as can be 85 parts by weight, 86 weight
Part, 87 parts by weight, 88 parts by weight, 89 parts by weight, 90 parts by weight, 91 parts by weight, 92 parts by weight, 93 parts by weight, 94 parts by weight or
95 parts by weight.
According to the present invention, the preservation liquid includes following component in parts by weight：
Physiological saline 6-10 parts by weight
Human serum albumin 4-6 parts by weight
Multiple electrolytes injection 85-90 parts by weight.
As most preferred technique scheme, the preservation liquid includes following component in parts by weight：
9 parts by weight of physiological saline
4 parts by weight of human serum albumin
87 parts by weight of Multiple electrolytes injection.
Second aspect, it is dry thin for preserving umbilical cord mesenchyma that the present invention provides preservation liquid composition as described in relation to the first aspect
The purposes of born of the same parents.
According to the present invention, a concentration of the 1 × 10 of the mescenchymal stem cell5-1×107/ ml, such as can be 1 × 105/
ml、2×105/ml、3×105/ml、4×105/ml、5×105/ml、6×105/ml、7×105/ml、8×105/ml、9×105/
ml、10×105/ml、20×105/ml、30×105/ml、40×105/ml、50×105/ml、60×105/ml、70×105/
ml、80×105/ml、90×105/ ml or 100 × 105/ ml, preferably 5 × 105-5×106/ ml, further preferably 2 ×
106/ml。
The third aspect, a kind of umbilical cord mesenchymal stem cells of the present invention preserve preparation, including preservation as described in relation to the first aspect
Liquid composition.
According to the present invention, it is that umbilical cord mesenchymal stem cells preserve liquid that the umbilical cord mesenchymal stem cells, which preserve preparation,.
Fourth aspect, the present invention provides a kind of store method of umbilical cord mesenchymal stem cells, using as described in the third aspect
Umbilical cord mesenchymal stem cells preserve preparation and preserve the umbilical cord mesenchymal stem cells.
According to the present invention, the store method of the umbilical cord mesenchymal stem cells includes the following steps：
(1) cell is collected and is counted；
(2) store method is specially and takes umbilical cord to preserve preparation with the umbilical cord mesenchymal stem cells to mix, and is placed in training
It supports and is cultivated in case.
According to the present invention, the temperature of step (2) described incubator is 35-40 DEG C, for example, can be 35 DEG C, 36 DEG C, 37 DEG C,
38 DEG C, 39 DEG C or 40 DEG C, preferably 36-38 DEG C.
According to the present invention, the CO of step (2) described incubator2A concentration of 3-8%, such as can be 3%, 4%, 5%,
6%, 7% or 8%, preferably 4-6%.
Compared with prior art, the present invention at least has the advantages that：
(1) the stem cell vigor after the present invention is preserved by preserving liquid is high, for 24 hours after stem cell vigor still 80% or more,
And the adherent ability of stem cell is good, and large effect is not generated to Stem cell surface marker after preserving, it is still dry thin for mesenchyma
Born of the same parents；
(2) preservation liquid composition components of the invention are simple, and umbilical cord mesenchymal stem cells is suitble to preserve at normal temperatures, prepare
Process is simple.
Figure of description
Fig. 1 (a) is after umbilical cord mesenchymal stem cells preserve 6h using physiological saline, and 96 orifice plates are inoculated with cellular morphology after 6h,
Fig. 1 (b) is after umbilical cord mesenchymal stem cells preserve 6h using preservation liquid, and 96 orifice plates are inoculated with cellular morphology after 6h, and Fig. 1 (c) is navel
After band mescenchymal stem cell preserves 6h using physiological saline, cellular morphology after the inoculation for 24 hours of 96 orifice plates, Fig. 1 (d) is umbilical cord mesenchyma
After stem cell is using liquid preservation 6h is preserved, cellular morphology after the inoculation for 24 hours of 96 orifice plates, Fig. 1 (e) uses for umbilical cord mesenchymal stem cells
After physiological saline preserves 6h, 96 orifice plates are inoculated with cellular morphology after 30h, and Fig. 1 (f) is that umbilical cord mesenchymal stem cells are protected using liquid is preserved
After depositing 6h, 96 orifice plates are inoculated with cellular morphology after 30h；
Fig. 2 (a) is after umbilical cord mesenchymal stem cells are preserved for 24 hours using physiological saline, and 96 orifice plates are inoculated with cellular morphology after 6h,
Fig. 2 (b) is after umbilical cord mesenchymal stem cells are preserved for 24 hours using preservation liquid, and 96 orifice plates are inoculated with cellular morphology after 6h, and Fig. 2 (c) is navel
After band mescenchymal stem cell is preserved for 24 hours using physiological saline, cellular morphology after the inoculation for 24 hours of 96 orifice plates, Fig. 2 (d) fills between umbilical cord
After matter stem cell is using liquid preservation for 24 hours is preserved, cellular morphology after the inoculation for 24 hours of 96 orifice plates, Fig. 2 (e) is umbilical cord mesenchymal stem cells
After being preserved for 24 hours using physiological saline, 96 orifice plates are inoculated with cellular morphology after 30h, and Fig. 2 (f) is umbilical cord mesenchymal stem cells using guarantor
After liquid storage preserves for 24 hours, 96 orifice plates are inoculated with cellular morphology after 30h.
Specific implementation mode
Of the invention for ease of understanding, it is as follows that the present invention enumerates embodiment.Those skilled in the art are it will be clearly understood that the implementation
Example is only to aid in the understanding present invention, should not be regarded as a specific limitation of the invention.
Experiment material
50ml centrifuge tubes (healthy and free from worry), 0.22um detachable needle syringes (PALL), 60ml syringes (BOON), Dispette
(Axygen), superclean bench (Su Jing is safe and sound), adjustable pipette (Biohit), ordinary optical microscope (Olympus), blood
Ball count version (QIUJING), countess calculating instruments (GIBCO companies)
Cell is P2 for cell, human serum albumin (the blue biology of China), Multiple electrolytes injection (Shanghai Bai Te), physiology salt
Water (the general Ji in Dongguan)
Embodiment 1 preserves the preparation of liquid (I)
The present embodiment provides a kind of preservation liquid, the preservation liquid includes following component in parts by weight：
9 parts by weight of physiological saline
4 parts by weight of human serum albumin
87 parts by weight of Multiple electrolytes injection；
The preparation method for preserving liquid, includes the following steps：
By formula ratio by after physiological saline, human serum albumin and Multiple electrolytes injection mixing, filtering is divided in centrifugation
Guan Zhong, packing preserve.
Embodiment 2 preserves the preparation of liquid (II)
The present embodiment provides a kind of preservation liquid, the preservation liquid includes following component in parts by weight：
6 parts by weight of physiological saline
6 parts by weight of human serum albumin
90 parts by weight of Multiple electrolytes injection；
The preparation method is the same as that of Example 1.
Embodiment 3 preserves the preparation of liquid (III)
The present embodiment provides a kind of preservation liquid, the preservation liquid includes following component in parts by weight：
10 parts by weight of physiological saline
4 parts by weight of human serum albumin
85 parts by weight of Multiple electrolytes injection；
The preparation method is the same as that of Example 1.
Embodiment 4 preserves the preparation of liquid (IV)
The present embodiment provides a kind of preservation liquid, the preservation liquid includes following component in parts by weight：
5 parts by weight of physiological saline
6 parts by weight of human serum albumin
95 parts by weight of Multiple electrolytes injection；
The preparation method is the same as that of Example 1.
Embodiment 5 preserves the preparation of liquid (V)
The present embodiment provides a kind of preservation liquid, the preservation liquid includes following component in parts by weight：
15 parts by weight of physiological saline
3 parts by weight of human serum albumin
80 parts by weight of Multiple electrolytes injection；
The preparation method is the same as that of Example 1.
Comparative example 1
Liquid is preserved only with physiological saline, other are same as Example 1.
Comparative example 2
Compared with Example 1, other same as Example 1 in addition to there is no human serum albumin.
Comparative example 3
Compared with Example 1, other same as Example 1 in addition to there is no Multiple electrolytes injection.
Comparative example 4
Compared with Example 1, other same as Example 1 in addition to the parts by weight of human serum albumin are 8 parts by weight.
Comparative example 5
Compared with Example 1, other same as Example 1 in addition to the parts by weight of physiological saline are 3 parts by weight.
Comparative example 6
Compared with Example 1, other same as Example 1 in addition to the parts by weight of physiological saline are 18 parts by weight.
Cell performance test
1) cell viability is tested
Specific cell performance test, includes the following steps：
(1) cell is collected：
1) prepare 25-30 bottles of T75 and P3 cells in advance, culture solution is poured into centrifuge tube, centrifuge 2000rpm/3min, it is past
The physiological saline of 10ml is added in culture bottle, washs dead cell and culture solution residue in culture bottle, is abandoned after washing；
2) physiological saline of the 0.25% pancreatin+1ml of 1ml is added into culture bottle, keeps flat culture bottle at once, shakes bottle
Son allows pancreatin to spread even and fine born of the same parents face；It takes a bottle to be capped, is put into microscopically observation digestion situation, when seeing that cell has been rounded
, digestion is terminated at once, and the supernatant after old medium centrifugal is poured into culture bottle；It is laid flat bottle, allows terminate liquid to be paved with as possible
Cell face, double swerve terminate digestion, and bottle standing is come, cell is blown and beaten with pipette, cell is all blown down as possible
Come, especially corner, action is soft, avoids generating bubble, collect in cell suspension to centrifuge tube, centrifuges 1000rpm/
10min；
3) remove supernatant after centrifuging, the physiological saline of 5ml be added, cell is blown even, add physiological saline to 40ml, blow it is even after
And draw 1ml countings；
4) 1000rpm/10min is centrifuged, after centrifugation, abandons supernatant, it is thin that a small amount of preservation liquid/physiological saline suspension is added
Born of the same parents, difference constant volume to 4.5ml；Cell suspension is dispensed into the EP pipes of sterilizing, each EP pipes fill 100 μ l, cell density 1 ×
106, mark；The cell that each time point is further cultured for will seal up sealed membrane；By EP pipes in different boxes.
(2) cell count
1) corresponding cell is taken out to time point, first first blows and beats 8 mixings with the rifle of 100 μ l, then toward in EP pipes plus
The physiological saline for entering 900 μ l is blown and beaten 10 times with the rifle of 1000 μ l, in the placenta basket for drawing 20 μ l to PE gloves, is mixed well thin
Born of the same parents 8~10 times；
2) it takes the tiling of PE gloves on the table, takes the trypan blue point of 20 μ l on gloves；The method for taking trypan blue：Platform is taken to expect
Before basket should not mixing, and only draw part above, when absorption draws the centre of working part as possible；
3) cell for drawing 10 μ l mixings, enters coverslip edge with tally at oblique 45 degree of subscripts；Another side method is the same,
Add simultaneously；
4) pay attention to when counting：Number is reached the standard grade and left line, does not count offline and right line, the Writing method of counting is per big lattice：
The time of viable count-dead cell number, counting is controlled as possible in 3~4min.
(3) continued to cultivate cell with 96 orifice plates
1) after preserving the half an hour that liquid fixed point counts, while the corresponding cell of physiological saline is taken, preparation continues to cultivate；
2) prepare two pieces of 96 orifice plates every time；
3) physiological saline of 400 μ l is added into per EP pipes in the case where grasping net platform, is diluted to 500 μ l and takes 100 μ l to one pipes new
EP pipes in (altogether about 2 × 105A cell) add the culture solution of 400 μ l to be diluted to 500 μ l (every 100 μ l/40000 cells), it takes
25 μ l are to per hole；
4) cell of each EP pipe adds 4 samples, side to add a blank control, two plate loading methods on each plate
Equally；
5) add after sample side marker it is good which side be to preserve liquid which side is that physiological saline, side sample time point and MTT add
The sample time is finally placed in incubator 37 DEG C, 5%CO2(lower layer being placed on as possible, close to the place of water) is cultivated.
Preservation liquid in embodiment 1-5 and comparative example 1-6 is subjected to cell preservation, 0,6,12,20, for 24 hours respectively to thin
Born of the same parents count, and calculate average viability, the results are shown in Table 1, specific as follows：
Table 1
Conspicuousness p<0.05
As it can be seen from table 1 embodiment 1-5 is after preserving cell for 24 hours, cell viability still has 80% or more, and from implementation
The comparison of example 1-5 finds out that the effect that the embodiment of the present application 1 preserves liquid is best, and cell viability is 83.8% after preserving for 24 hours；
Find out from embodiment and comparative example, comparative example preserves cell vigor after 12h and declines comparatively fast, and rear cell viability only stays for 24 hours
It is even lower to deposit cell viability 50%.
2) adherent experiment
(1) corresponding 96 orifice plate is taken out from incubator, and WST-1 reagents are taken out from refrigerator；
(2) use lancet that the reagent of the WST-1 of 10ul is added into the hole that each is loaded；
(3) tag time is put into incubator and continues culture 4 hours；
(4) proliferation activity is surveyed, i.e., two groups of samples of each period are inoculated with simultaneously in 96 orifice plates (2 pieces), respectively at training totally
After WST-1 reagents incubation 4h is added after foster 6h and 36h, observation is inoculated in the cell in T25 culture bottles, and comparing embodiment 1 (preserves
Liquid group) and comparative example 1 (physiological saline group) group in cell adherent proliferative conditions, as a result such as Fig. 1 (a)-Fig. 1 (f) and Fig. 2 (a)-
Shown in Fig. 2 (f)；
From Fig. 1 (a)-Fig. 1 (f) and Fig. 2 (a)-Fig. 2 (f) as can be seen that either culture 6h still for 24 hours after, embodiment 1
The adherent situation of cell be better than comparative example 1.
3) drain cell detects
After 1 group of embodiment 1 and comparative example are preserved 20h at 4 DEG C, it is carried out at the same time FCM analysis, observation length
Whether phase preservation has an impact the expression of cell surface marker, and the results are shown in Table 2：
Table 2
From table 2 it can be seen that the CD73 in embodiment and comparative example, CD90, CD105 expression in 95% or more, CD14,
CD45, CD34, HLA-DR expression are 2% hereinafter, cell of the explanation after embodiment and comparative example preserves liquid preservation is still
Mescenchymal stem cell.
In conclusion the present invention is high by the stem cell vigor after preserving liquid preservation, rear stem cell vigor is still 80% for 24 hours
More than, and the adherent ability of cell is good, and large effect is not generated to Stem cell surface marker after preserving, it is still dry for mesenchyma
Cell.
Applicant states that the present invention illustrates detailed process equipment and the technological process of the present invention by above-described embodiment,
But the invention is not limited in above-mentioned detailed process equipment and technological processes, that is, it is above-mentioned detailed not mean that the present invention has to rely on
Process equipment and technological process could be implemented.Person of ordinary skill in the field it will be clearly understood that any improvement in the present invention,
The addition of equivalence replacement and auxiliary element to each raw material of product of the present invention, the selection etc. of concrete mode all fall within the present invention's
Within protection domain and the open scope.
Claims (10)
1. a kind of preservation liquid composition, which is characterized in that the preservation liquid includes following component in parts by weight：
Physiological saline 5-15 parts by weight
Human serum albumin 3-6 parts by weight
Multiple electrolytes injection 80-95 parts by weight.
2. preserving liquid composition as described in claim 1, which is characterized in that the preservation liquid includes such as the following group in parts by weight
Point：
Physiological saline 6-10 parts by weight
Human serum albumin 4-6 parts by weight
Multiple electrolytes injection 85-90 parts by weight.
3. preserving liquid composition as claimed in claim 1 or 2, which is characterized in that the preservation liquid includes such as in parts by weight
Lower component：
9 parts by weight of physiological saline
4 parts by weight of human serum albumin
87 parts by weight of Multiple electrolytes injection.
4. the preservation liquid composition as described in one of claim 1-3 is used to preserve the purposes of umbilical cord mesenchymal stem cells.
5. purposes as claimed in claim 4, which is characterized in that a concentration of the 1 × 10 of the mescenchymal stem cell5-1×107/
Ml, preferably 5 × 105-5×106/ ml, further preferably 2 × 106/ml。
6. a kind of umbilical cord mesenchymal stem cells preserve preparation, which is characterized in that including as claimed in any one of claims 1-3
Preserve liquid composition；
Preferably, it is that umbilical cord mesenchymal stem cells preserve liquid that the umbilical cord mesenchymal stem cells, which preserve preparation,.
7. a kind of store method of umbilical cord mesenchymal stem cells, which is characterized in that filled between use umbilical cord as claimed in claim 6
Matter stem cell preserves preparation and preserves the umbilical cord mesenchymal stem cells.
8. store method as claimed in claim 7, which is characterized in that include the following steps：
(1) cell is collected and is counted；
(2) store method is specially and takes umbilical cord to preserve preparation with the umbilical cord mesenchymal stem cells to mix, and is placed in incubator
Middle culture.
9. store method as claimed in claim 7 or 8, which is characterized in that the temperature of step (2) described incubator is 35-40
DEG C, preferably 36-38 DEG C.
10. the store method as described in one of claim 7-9, which is characterized in that the CO of step (2) described incubator2Concentration
For 3-8%, preferably 4-6%.
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