CN101990888B - Stem cell freezing medium and freezing method of stem cells - Google Patents

Abstract

The invention provides a stem cell freezing medium and a freezing method of stem cells. The freezing medium contains dimethyl sulfoxide and hydroxyethyl starch and has great clinical application value. The invention also provides the freezing method of stem cells.The stem cell freezing medium in the invention is adopted by the method.In the invention,the cells frozen with the stem cell freezing medium has the advantage of high recovery/survival rate.

Description

The cryopreservation methods of stem cell cryopreserving liquid and stem cell

Technical field

The present invention relates to the cryopreservation methods of stem cell cryopreserving liquid and stem cell.

Background technology

Frozen process can significantly change thermodynamics, chemistry and the physical environment of cell, has and follows the danger that causes biological injury.The variation of temperature depends primarily on cooling or the caused heat conducting final condition of temperature-rising method, is also subject to cell to melt again the impact of latent heat effect in process.Minimum for the damage of cell in frozen-thaw process is down to, must further optimize chemistry and temperature operation process.But need to before cooling, add one or both cryoprotective agents, after dissolving again by its removal.

Survival rate is the highest when making cell recovery, and best freezing conditions is to reduce as much as possible intracellular crystal formationly, reduces the low temperature injury that in cell, the solid formed high concentrations of solutes of water-setting causes cell.Above-mentioned requirements can obtain by following method: (1) is slowly freezing, makes ICW leave cell, but can not be too slow, to avoid ice crystal to form; (2) with hydrophilic cryoprotective agent, get rid of moisture; (3) Cell protection at alap temperature, reduces the impact of high salt concentration on protein denaturation in ice; (4) rapid fluid resuscitation, reduce in cell crystal formation, and the generation of soluble components when remaining ice dissolves in cell.

Frozen influence factor: cell concn: with higher concentration freeze-stored cell, the recovery motility rate of cell is higher.Freezing rate: most of cultured cells are the highest with the speed freeze survival rate of 1 ℃/min, this may be that quick freezing reduces ice crystal formation and slow freezing increases a compromise between ECW migration.

Frozen storing liquid: cell suspension carries out freezing conventionally after adding cytoprotective glycerine or dimethyl sulfoxide (DMSO) (DMSO), the latter's better effects if, reason may be relevant by force compared with glycerine with the ability of its penetration cell.Working concentration is 5%-15%.The cytoprotective of commonly using also has, hydroxyethylamyle (HES), polyvinylpyrrolidone (PVP), polyoxyethylene glycol (PEG) etc.

The reports such as Japanese scholars Makino S, adopt the 6% hydroxyethylamyle associating frozen autologous peripheral blood stem cells of 5%DMSO (PBSCs), than simple 10%DMSO cryopreservation methods better effects if, the method by cell cryopreservation in-80 ℃, can obtain higher recovery motility rate (88.4+/-3.6%), and after recovery, cell keeps hematopoiesis active; Reach the frozen of 1-5 and also can obtain more satisfied effect; After the autologous PBSCs of 10 patients with hematological tumor infusions recovery, all showed cell is successfully implanted, wherein 7 patients obtain that symptom alleviates completely for up to 3.5-15 month.The method is widely adopted in Japan.

The reports such as Spain scholar Servei D ' Hematologia, adopt 10%DMSO and the frozen autologous PBSCs of autologous plasma and medullary cell, recovery motility rate can reach 90%, after the autologous PBSCs of 11 patients with hematological tumor infusion recoveries, all showed cell is implanted, wherein 10 patients obtain that symptom alleviates completely for up to 3-9 month.

Norway's scholar's research finds, with respect to 10%DMSO, PBSCs apoptosis and mortality ratio that 5%DMSO is frozen are all lower; 103 patients with hematological tumor infusion 10%DMSO and the frozen autologous PBSCs of 5%DMSO, its clinical effectiveness there is no significant difference.This scholar thinks, from the viewpoint of security, should select 5%DMSO.Holland scholar's research also shows, the frozen recovery motility rate of 5%DMSO is higher than 10%DMSO.

In sum, hemopoietic stem cell bank tends to adopt 5%DMSO, if can be equipped with autologous plasma, frozen effect is better.Meanwhile, we should be noted that, the frozen PBSCs of infusion DMSO may be relevant with some toxic reactions, such as vomiting, heart function imbalance, allergy and acute renal failure, so guaranteeing, under the prerequisite of frozen effect, should to reduce the concentration of DMSO as far as possible.

Summary of the invention

For helping, understand the present invention, defined some terms below.Term defined herein has the common implication of understanding of those of ordinary skill in field of the present invention.

Unless otherwise indicated, the percentage composition of HES represents weight percentage herein, and other percentage compositions all represent volumn concentration.

Unless otherwise indicated, " basic medium " herein refers to and contains the substratum that Growth of Cells is bred required basic nutrition material, can be for most cells growth.

Unless otherwise indicated, DMSO in the present invention is that the English of dimethyl sulfoxide (DMSO) is called for short, HES is that the English of hydroxyethylamyle is called for short, AB represents people AB serum, FBS is that the English of foetal calf serum is called for short, MM is that the English of basic medium is called for short, and HAS behaves sero-abluminous English abbreviation, and the English that MSC is umbilical cord mesenchymal stem cells is called for short.

An object of the present invention is to provide a kind of stem cell cryopreserving liquid, this frozen storing liquid can make frozen stem cell have good frozen resurrection rate, avoids containing animal serum and substratum simultaneously, makes this frozen storing liquid have more clinically practical value.

Another object of the present invention has been to provide a kind of method of stem cell cryopreserving recovery, and the method adopts stem cell cryopreserving liquid of the present invention.

One aspect of the present invention provides a kind of stem cell cryopreserving liquid, and wherein said frozen storing liquid contains dimethyl sulfoxide (DMSO) and hydroxyethylamyle.

Preferably, the dimethyl sulfoxide (DMSO) that contains 5-10% volume in described frozen storing liquid and the hydroxyethylamyle of 3-6% weight.

More preferably, the dimethyl sulfoxide (DMSO) that described frozen storing liquid contains 5% volume and the hydroxyethylamyle of 6% weight.

Preferably, described frozen storing liquid also contains people AB serum.

More preferably, the content of described people AB serum is 10-80% volume.

Again preferably, the content of described people AB serum is 10-50% volume.

The people AB serum of the dimethyl sulfoxide (DMSO) that most preferably, described frozen storing liquid contains 5% volume, the hydroxyethylamyle of 5% weight and 10% volume.

The people AB serum of the dimethyl sulfoxide (DMSO) that preferably, described frozen storing liquid contains 5% volume, the hydroxyethylamyle of 3% weight and 45% volume.

Preferably, described frozen storing liquid does not contain people AB serum.

Further aspect of the present invention also provides a kind of stem cell cryopreserving method, and the method comprises the following steps: a. adds frozen storing liquid of the present invention by postdigestive stem cell, obtains cell suspending liquid;

B. described cell suspending liquid is freezing, then the cell suspending liquid after freezing is moved in liquid nitrogen and preserved.

Preferably, the cell number in the cell suspending liquid in wherein said cell suspending liquid is 1*10 ⁶-4*10 ⁷individual/ml.

Beneficial effect of the present invention is: adopt the cell recovery motility rate in 4 generations of passage of dimethyl sulfoxide (DMSO), the hydroxyethylamyle of 3-6% weight and the people AB serum of 10-80% volume that frozen storing liquid of the present invention contains 5-10% volume within the scope of 60%-85%, the cell recovery motility rate of dimethyl sulfoxide (DMSO), the hydroxyethylamyle of 3-5% weight and the people AB serum of 10-45% volume that preferably frozen storing liquid contains 5% volume can reach 90%.Cell after recovery is still highly expressed umbilical cord mesenchymal stem cells surface antigen.Induction Analytical Chemical Experiment shows, after umbilical cord mesenchymal stem cells recovery, can induce as becoming fat, scleroblast.

Accompanying drawing explanation

Fig. 1 shows the dimethyl sulfoxide (DMSO) frozen storing liquid recovery cellular form figure after 1 day, and wherein Fig. 1-a indicated concentration is the dimethyl sulfoxide (DMSO) frozen storing liquid recovery of the 5% volume cellular form figure after 1 day; Fig. 1-b indicated concentration is the cellular form figure after the dimethyl sulfoxide (DMSO) frozen storing liquid of 8% volume is recovered 1 day; Fig. 1-c indicated concentration is the cellular form figure after the dimethyl sulfoxide (DMSO) frozen storing liquid of 10% volume is recovered 1 day.

Fig. 2 shows after the cell recovery under different frozen schemes the state photograph of the 1st day.The cellular form figure of the AB frozen storing liquid recovery of HES+10% volume of DMSO+5% weight that wherein Fig. 2-a represents 5% volume after 1 day; The cellular form figure of the AB frozen storing liquid recovery of HES+45% volume of DMSO+3% weight that Fig. 2-b represents 5% volume after 1 day; The cellular form figure of the MM frozen storing liquid recovery of FBS+80% volume of DMSO+10% volume that Fig. 2-c represents 10% volume after 1 day.

Fig. 3 shows the expression of surface antigen of AB frozen storing liquid recovery cell of HES+10% volume of the DMSO+5% weight of flow cytometer detection 5% volume, carries out cell counting.Wherein Fig. 3-a represents to use CD ₇₃detect the expression of surface antigen, carry out cell counting; Fig. 3-b represents to use CD ₉₀detect the expression of surface antigen, carry out cell counting; Fig. 3-c represents to use CD ₃₄detect the expression of surface antigen, carry out cell counting; Fig. 3-d represents to use CD ₁₀₅detect the expression of surface antigen, carry out cell counting; Fig. 3-e represents to use CD ₄₅detect the expression of surface antigen, carry out cell counting.

Fig. 4 shows the expression of surface antigen of AB frozen storing liquid recovery cell of HES+45% volume of the DMSO+3% weight of flow cytometer detection 5% volume, carries out cell counting.Wherein Fig. 4-a represents to use CD ₇₃detect the expression of surface antigen, carry out cell counting; Fig. 4-b represents to use CD ₉₀detect the expression of surface antigen, carry out cell counting; Fig. 4-c represents to use CD ₃₄detect the expression of surface antigen, carry out cell counting; Wherein Fig. 4-d represents to use CD ₁₀₅detect the expression of surface antigen, carry out cell counting; Fig. 4-e represents to use CD ₄₅detect the expression of surface antigen, carry out cell counting.

Fig. 5 shows the surface antigen of MM frozen storing liquid recovery cell of FBS+80% volume of the DMSO+10% volume of flow cytometer detection 10% volume, and wherein Fig. 5-a represents to use CD ₇₃detect the expression of surface antigen, carry out cell counting; Fig. 5-b represents to use CD ₉₀detect the expression of surface antigen, carry out cell counting; Fig. 5-c represents to use CD ₁₀₅detect the expression of surface antigen, carry out cell counting; Wherein Fig. 5-d represents to use CD ₃₄detect the expression of surface antigen, carry out cell counting; Fig. 5-e represents to use CD ₄₅detect the expression of surface antigen, carry out cell counting.

Fig. 6 shows the induction Analytical Chemical Experiment of AB frozen storing liquid recovery cell of HES+10% volume of the DMSO+5% weight of 5% volume.Wherein Fig. 6-a represents to drip with oil red O stain showed cell lactones after becoming fat induction; Fig. 6-b represents that osteogenic induction hystazarin red colouring shows calcium tubercle.

Fig. 7 shows the induction Analytical Chemical Experiment of AB frozen storing liquid recovery cell of HES+45% volume of the DMSO+3% weight of 5% volume.Wherein Fig. 7-a represents to drip with oil red O stain showed cell lactones after becoming fat induction; Fig. 7-b represents that osteogenic induction hystazarin red colouring shows calcium tubercle.

Fig. 8 shows the induction Analytical Chemical Experiment of MM frozen storing liquid recovery cell of FBS+80% volume of the DMSO+10% volume of 10% volume.Wherein Fig. 8-a represents to drip with oil red O stain showed cell lactones after becoming fat induction; Fig. 8-b represents that osteogenic induction hystazarin red colouring shows calcium tubercle.

Embodiment

Following examples are used for explaining the present invention, and are not used in restriction the present invention.

the impact of DMSO concentration on frozen effect in embodiment 1, research frozen storing liquid

(1) first stage experiment

Experiment purpose

The impact of DMSO concentration on frozen effect in research frozen storing liquid.

Experimental technique

Set up 5%, 8%, 10% 3 DMSO concentration, in conjunction with perfect medium, form frozen storing liquid, after peptic cell, add frozen storing liquid according to a conventional method, be placed in freezing storing box, move into-80 ℃ of refrigerators, move in liquid nitrogen next day.After one week, recover, relatively cell viability and cell state.

Experimental result

(1) respectively organize cell viability without significant difference.

(2) respectively organize cell state also without significant difference.Cellular form is short fusiformis, is evenly distributed, in good condition, sees Fig. 1-a, Fig. 1-b and Fig. 1-c.After 3 days, cell degree of converging reaches more than 80%.

Experiment conclusion

5%, 8%, 10% 3 DMSO concentration does not have notable difference to the frozen effect of umbilical cord mesenchymal stem cells (being called for short MSC).

embodiment 2, change HES final concentration, develop into quality than final concentration by volume ratio final concentration, and visit beg for other feasible cryopreservation methods.

Experiment purpose: change HES final concentration, develop into quality than final concentration by volume ratio final concentration, and inquire into other feasible cryopreservation methods.

Experimental technique: with embodiment 1

Experimental result:

1. inquire into the frozen effect of following scheme: AB is people AB serum; MM is basic medium; FBS is foetal calf serum; HES is hydroxyethylamyle, and following concentration HES is quality final concentration, and all the other are volume final concentration.

Table 1

	5％DMSO +5％HES +10％AB	5％DMSO +3％HES +45％AB	10％DMSO +10％FBS +80％MM
				NO 1	90.41％	89.10％	89.96％
NO 2	91.38％	90.94％	90.01％
				NO 3	82.03％	83.34％	86.29％
NO 4	89.41％	89.74％	90.81％
				NO 5	89.55％	89.20％	90.96％
NO 6	88.62％	89.25％	89.70％
				On average	88.57％± 0.11％	88.60％± 0.07％	89.62％± 0.03％

Completely randomized design data one-way analysis of variance, each group difference not statistically significant.Recovery is cultivated rear first day and is observed discovery, and cell state is good, and after 3 days, cell degree of converging reaches more than 85%.Flow cytometer detection result shows, cell is still highly expressed MSC surface antigen.Induction Analytical Chemical Experiment shows, after umbilical cord MSC recovery, can induce as becoming fat, scleroblast.

After each scheme recovery, the cell state figure of the 1st day is shown in Fig. 2.Scheme 5%DMSO+5%HES+10%AB flow cytometer detection the results are shown in Figure 3.

Scheme 5%DMSO+3%HES+45%AB flow cytometer detection the results are shown in Figure 4.

Scheme 10%DMSO+10%FBS+80%MM flow cytometer detection the results are shown in Figure 5.

Scheme 5%DMSO+5%HES+10%AB becomes fat, Osteoinductive differentiation coloration result to see Fig. 6.

Scheme 5%DMSO+3%HES+45%AB becomes fat, Osteoinductive differentiation coloration result to see Fig. 7.

Scheme 10%DMSO+10%FBS+80%MM becomes fat, Osteoinductive differentiation coloration result to see Fig. 8.

Each surface antigen expression rate of flow cytometer detection is as following table 2

Table 2

	CD 73	CD 90	CD 105	CD34	CD 45
						5％DMSO+5％HES+10％AB	98.5％	99.1％	96.8％	0.7％	0.1％
5％DMSO+3％HES+45％AB	98.1％	99.2％	95.6％	0.1％	0.1％
						10％DMSO+10％FBS+80％MM	97.8％	98.7％	96.9％	0.2％	0.2％

Above-mentioned flow cytometer detection result shows, the cell of this two prescriptions case recovery is still highly expressed MSC surface antigen.

2. density experiment:

The cell of the different densities that the frozen scheme of AB of the HES+10% volume of the DMSO+5% weight of approach and application 5% volume is frozen, the difference of its recovery motility rate.Recovery motility rate is as following table 3

Table 3

	1×10 6/ml	5×10 6/ml	1×10 7/ml	4×10 7/ml
					NO 1	92.0％	94.5％	94.0％	97.5％
NO 2	94.0％	95.0％	95.0％	91.5％
					NO 3	96.5％	97.0％	97.0％	94.5％
NO 4	94.0％	95.0％	97.5％
					NO 5		97.5％
NO 6	93.5％	95.5％	88.5％	95.5％
					NO 7		97.0％	93.5％	94.0％
NO 8			96.5％
					Mean value	94±1.6％	95.9± 1.2％	94.6± 3.1％	94.6± 2.2％

Frozen scheme: the AB of the HES+10% volume of the DMSO+5% weight of 5% volume

Completely randomized design data one-way analysis of variance, each group difference not statistically significant, P=0.466.Average 94.9 ± 2.2%

3. washings experiment

The cell that the different washings washing of approach and application has just been thawed, the difference of its recovery motility rate.Recovery motility rate is as following table 4

Table 4

	Cold saline	Cold HES injection liquid	Cold DMEM/F-12
				NO 1	91.0％	95.0％	94.0％
NO 2	91.5％	94.0％	91.5％

NO 3	95.5％	95.5％	96.5％
				NO 4	95.5％	96.0％	95.0％
NO 5	96.0％	94.0％	97.0％
				NO 6	91.5％	97.0％	95.5％
Mean value	93.5±2.4％	95.3±1.2％	94.9±2.0％

Frozen scheme: the AB completely randomized design data one-way analysis of variance of the HES+10% volume of the DMSO+5% weight of 5% volume, each group difference not statistically significant.

Experiment conclusion:

Above-mentioned each experimental program is all obtained good frozen effect;

Density experiment shows, 1 * 10 ⁶/ ml to 4 * 10 ⁷between/ml, each density does not have a significant effect to frozen effect;

Washings experiment shows, with above-mentioned three kinds of cold washingss, cell recovery motility rate is not had a significant effect.

Claims (7)

1. a stem cell cryopreserving liquid, the people AB serum of the hydroxyethylamyle of the dimethyl sulfoxide (DMSO) that described frozen storing liquid contains 5-10% volume, 3-6% weight and 10%-80% volume.

2. stem cell cryopreserving liquid according to claim 1, the hydroxyethylamyle of the dimethyl sulfoxide (DMSO) that described frozen storing liquid contains 5% volume and 6% weight.

3. stem cell cryopreserving liquid according to claim 1 and 2, the content of wherein said people AB serum is 10-50% volume.

4. stem cell cryopreserving liquid according to claim 1, the people AB serum of the hydroxyethylamyle of the dimethyl sulfoxide (DMSO) that wherein said frozen storing liquid contains 5% volume, 5% weight and 10% volume.

5. stem cell cryopreserving liquid according to claim 1, the people AB serum of the hydroxyethylamyle of the dimethyl sulfoxide (DMSO) that wherein said frozen storing liquid contains 5% volume, 3% weight and 45% volume.

6. a stem cell cryopreserving method, the method comprises the following steps:

A. postdigestive stem cell is added to the frozen storing liquid described in any one in claim 1-5, obtain cell suspending liquid;

B. described cell suspending liquid is freezing, then the cell suspending liquid after freezing is moved in liquid nitrogen and preserved.

7. method according to claim 6, the cell number in wherein said cell suspending liquid is 1 * 10 ⁶-4 * 10 ⁷individual/ml.

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