CN102273438A - Cryopreservation protection excipient for medicinal cell injection - Google Patents
Cryopreservation protection excipient for medicinal cell injection Download PDFInfo
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- CN102273438A CN102273438A CN2011102319836A CN201110231983A CN102273438A CN 102273438 A CN102273438 A CN 102273438A CN 2011102319836 A CN2011102319836 A CN 2011102319836A CN 201110231983 A CN201110231983 A CN 201110231983A CN 102273438 A CN102273438 A CN 102273438A
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Abstract
The invention provides a cryopreservation protection excipient for medicinal cell injection. The cryopreservation protection excipient for the medicinal cell injection comprises a certain amount of polyethylene glycol, propylene glycol, albumin and sodium chloride. The excipient can also be used as a protective agent for vitrified cryopreservation of biological tissue samples. The excipient can reduce the harm to cells and maintain cell viability in the cryopreservation and anabiosis process of the medicinal cell injection; and the medicinal cell injection containing the excipient can be subjected to vitrified cryopreservation at low temperature for a long time, can be directly used for cell transplantation after being thawed, and has the characteristics of safety, convenience, quickness and the like. In addition, the excipient also has the effects of supplementing nutrition, enhancing immunity and strengthening cell therapy effects, and is more suitable for cell therapy.
Description
Technical field
The present invention relates to biomedicine field; the auxiliary material of medicine particularly; the glass frozen preservation technology that also relates to biological products; be specifically related to a kind of frozen protection auxiliary material of medicinal cell injection; by in medicinal cell product, adding polyethylene glycol; 1; the 2-propane diols; albumin; frozen protectant such as sodium chloride is realized the glass frozen preservation of medicinal cell product; improve the anabiosis rate after thawing; and safety non-toxic; realize medicinal cell product thaw the back direct clinical use, this auxiliary material also has extra-nutrition in addition; enhancing immunity; strengthen the function of cell therapy effect.
Background technology
Cell therapy claims the living cells treatment again, is meant with living cells and repairs pathology or injured tissues.
The cell that uses in the cell therapy can have multiple source, according to the retrieval to national Bureau of Drugs Supervision website, the cell therapy product that present China enters clinical testing has mesenchymal stem cells MSCs, Cord blood megakaryoblast, Cord blood CFU-E, cytokine induced kill cell (CIK cell) etc.; According to " the allowing the 3rd class medical skill catalogue of clinical practice in the first batch " of Ministry of Public Health's promulgation in 2009, cellular transplantation therapy technology (except the stem cell), candidate stem cell treatment technology have been allowed to be applied to clinical.Also have various kinds of cell such as neural stem cell, embryonic stem cell, retinal pigment epithelium to enter clinical testing or animal experiment in addition both at home and abroad as medicine or medical skill.
Because cell therapy will use living cells, medicinal cell product must keep cell activity in storage process.Traditional long term storage method is to add about 10% DMSO as cryoprotective agent in cell product, and is frozen then in-196 ℃ of liquid nitrogen or-80 ℃ of ultra low temperature freezers.Yet DMSO has very high toxic and side effect, makes symptoms such as the individuality generation of accepting to transplant is felt sick, vomiting, stomachache usually, also can have side effects to cardiovascular, respiratory system, nervous centralis and kidney etc.When using DMSO as frozen dose, it is just safer to carry out cell transplantation again after the domestication of carrying out a period of time after the cell thawing recovery is cultivated.Therefore the frozen protectant of seeking that a kind of toxic and side effect is little, can directly transplanting behind the cell thawing is extremely important.
Method for vitrification is a kind of method of preserving biomaterial under complete vitrifying state.Vitrifying is meant that liquid state changes amorphous solidification process into, and normal molecule and ion distribution can be avoided the physical injury that biomaterial is caused because of freezing when glassy solids can keep liquid.Method for vitrification is simple to operate fast, and pair cell, tissue damage are little, are a kind of long-term preservation methods of very promising biomaterial.The invention provides a kind of frozen protection auxiliary material of new pharmaceutical grade cell injection, comprise polyethylene glycol, 1, compositions such as 2-propane diols, albumin, sodium chloride.The frozen protection auxiliary material of this cell injection can effectively form the long-term frozen effect of low temperature glassization of cell or tissue; and can directly transplanting after using the frozen cell injection of this auxiliary material to thaw; have characteristics such as safe, convenient, fast; have the function of extra-nutrition, enhancing immunity, enhancing cell therapy effect in addition, be more suitable for cell therapy.
Summary of the invention
The objective of the invention is to specific demand, a kind of frozen protection auxiliary material of medicinal cell injection is provided, make cell therapy safer, convenient, fast at cell therapy.
The present invention relates to a kind of medicinal biological organization sample, for example, the frozen protection auxiliary material of cell injection, it contains polyethylene glycol, 1,2-propane diols, albumin and sodium chloride composition.In specific embodiment, described polyethylene glycol is at described biological organization sample, and for example the final concentration in the cell injection is 5-100g/L, 25-100g/L, 50-100g/L, 75-100g/L, 80-100g/L, 90-100g/L; Described 1, the 2-propane diols is at described biological organization sample, and for example the final concentration in the cell injection is 50-400g/L, 100-400g/L, 150-400g/L, 200-400g/L, 250-400g/L, 300-400g/L; Described albumin is at described biological organization sample, and for example the final concentration in the cell injection is 10-100g/L, 30-100g/L, 50-100g/L, 70-100g/L, 80-100g/L, 90-100g/L; Described sodium chloride is at described biological organization sample, and for example the final concentration in the cell injection is 8.5-9.5g/L, 8.6-9.4g/L, 8.7-9.3g/L, 8.8-9.2g/L, 8.9-9.1g/L, 9g/L.More than listed polyethylene glycol, 1, the final concentration scope of 2-propane diols, albumin and sodium chloride composition can make up arbitrarily, is mixed with the frozen protection auxiliary material of medicinal cell injection of the present invention.For example, in one embodiment, described polyethylene glycol, 1,2-propane diols, albumin and the final concentration of sodium chloride composition in described cell injection are:
Polyethylene glycol (PEG), 5-100g/L;
1,2-propane diols, 50-400g/L;
Albumin, 10-100g/L;
Sodium chloride, 8.5-9.5g/L.
Preferably, described polyethylene glycol, 1,2-propane diols, albumin and the sodium chloride final concentration in described cell injection is:
Polyethylene glycol, 50-100g/L,
1, the 2-propane diols, 100-400g/L,
Albumin, 50-100g/L,
Sodium chloride, 8.5-9.5g/L.
More preferably, described polyethylene glycol, 1,2-propane diols, albumin and the sodium chloride final concentration in described cell injection is:
Polyethylene glycol, 80-100g/L,
1, the 2-propane diols, 200-400g/L,
Albumin, 80-100g/L,
Sodium chloride, 9g/L.
Prepare the described polyethylene glycol, 1 that frozen protection auxiliary material of the present invention is adopted, 2-propane diols, albumin, sodium chloride preferably are the pharmaceutical grade product; Described albumin is human serum albumin or be called human serum albumins preferably; Described polyethylene glycol preferably molecular weight is 400 polyethylene glycol (PEG400).The frozen protection auxiliary material of cell injection of the present invention can also be used as the protectant of biological organization sample glass frozen preservation as the frozen protectant of cell injection.
The present invention relates to the frozen protectant of a kind of biological organization sample, contain polyethylene glycol, 1,2-propane diols, albumin and sodium chloride composition.In one embodiment, the final concentration of described composition in described biological organization sample is respectively:
Polyethylene glycol (PEG), 5-100g/L;
1,2-propane diols, 50-400g/L;
Albumin, 10-100g/L;
Sodium chloride, 8.5-9.5g/L.
Preferably, the final concentration of described composition in described biological organization sample is:
Polyethylene glycol, 50-100g/L,
1, the 2-propane diols, 100-400g/L,
Albumin, 50-100g/L,
Sodium chloride, 8.5-9.5g/L.
More preferably, the final concentration of described composition in described biological organization sample is:
Polyethylene glycol, 80-100g/L,
1, the 2-propane diols, 200-400g/L,
Albumin, 80-100g/L,
Sodium chloride, 9g/L.
In above embodiment, described albumin is a human serum albumin; Described polyethylene glycol is that molecular weight is 400 polyethylene glycol (PEG400).Above-described biological organization sample is preferably cell suspension after the fresh separated that is used for cell transplantation or the cell suspension behind the cryopreservation resuscitation or the like.
Term " cell therapy " or be called " living cells treatment " is meant competent cell is injected in the body, stimulate degenerate, old and feeble, damaging cells, promote its renewal, reparation, thereby recover organ dysfunction.Theory according to the Ni Hansi certify by a physician, these cells begin circulation from the position of injection, even they identify to it and come the similar organ of source organ, and assemble thereon, for example, liver cell enters that liver, sexual cell move to sexual organ, heart cell enters heart or the like.Various kinds of cell such as mesenchymal stem cells MSCs, Cord blood megakaryoblast, neural stem cell, embryonic stem cell, retinal pigment epithelium have entered clinical testing or animal experiment as medicine both at home and abroad at present.In the clinical practice of living cells, it is vital keeping cell activity.The frozen protection auxiliary material of cell injection of the present invention can make the quantity of cell injection living cells after 90 hours and 30 days of redissolution still remain on more than 85%.Simultaneously, frozen protection auxiliary material of the present invention also is safe by the zoopery proof.
" vitrifying " is frozen to be meant in temperature-fall period, does not produce ice crystal, and the process that solution is solidified.It is different with the freezing process of common liquid twin crystal or partially crystallizable.In auxiliary material of the present invention; polyethylene glycol and albumin play the frozen protective effect of impermeability; can deep enough cell interior, have the freezing point of reduction, reduce that ice crystal forms, electrolytical concentration in the dilute solution, reduce solute damage, the inside and outside osmotic pressure of statocyte, alleviate effect such as cell shrink degree.1,2-propane diols and sodium chloride play the frozen protective effect of permeability.Mentioned component is united use, has produced significant synergy, forces some thermodynamic parameter that change has taken place, and reduces such as vitrification point rising, vitrifying desired concn, has improved preservation effect, has realized that the low temperature glassization of cell injection is frozen for a long time.
Each composition in the above-mentioned auxiliary material of the present invention all is common chemical reagent or biological products, wherein polyethylene glycol can be available from Qingdao Tian Liyuan bio tech ltd, 1, the 2-propane diols can be available from Jinhua Chemical (Group) Corp., Ltd., albumin can be available from the Shanghai institute of Biological Products, and the form that sodium chloride can physiological saline is available from Jilin nation's medicine company limited company all.
Embodiment
(1) configuration auxiliary material: with polyethylene glycol, 1,2-propane diols, albumin, each adjunct ingredient of sodium chloride add in the water according to aforementioned concentration and dissolve, perhaps with polyethylene glycol, 1,2-propane diols, each adjunct ingredient of albumin add in the physiological saline according to aforementioned concentration and dissolve, and make auxiliary material solution;
(2) preparation parenteral solution: be put in the centrifuge tube cell in the culture fluid centrifugal, abandon supernatant, the auxiliary material solution that step (1) is prepared adds in the centrifuge tube re-suspended cell to, make the concentration of cell in solution reach 1.0 * 10^6 cell/ml, make medicinal cell injection;
(3) frozen: that the medicinal cell injection of step (2) the preparation step according to " 4 ℃ 1 hour →-20 ℃ 1 hour →-70 ℃ 10 hours → liquid nitrogen long term storages " is stored in the liquid nitrogen;
(4) redissolve: before the use, the medicinal cell injection that stores in the step (3) is taken out from liquid nitrogen, be positioned in 35 ℃ and redissolve, be used for cell transplantation then.
Embodiment 1:
(1) according to following recipe configuration 100ml auxiliary material solution:
(2) get human nerve stem cell (source: from the Cord blood that Shanghai Blood Center umbilical cord blood bank gives, extract mescenchymal stem cell, adding growth factor EGF and each 20ng/ml of bFGF induces differentiation to become neural stem cell) 1.0 * 10^8, the centrifugal supernatant of abandoning, it is resuspended to add 100ml auxiliary material solution, through automated cell calculating instrument counting, cell concentration reaches 1.0 * 10^6/ml, makes cell injection, divide to install to 10 frozen blood bags of 50ml, every bag of amount of viable cell is 1.0 * 10^7.
(3) put into 4 ℃ 1 hour ,-20 ℃ 1 hour ,-70 ℃ 10 hours, change in the liquid nitrogen and store.
(4) respectively take out 5 bags of cell injections, 35 ℃ of redissolution respectively at the liquid nitrogen storage after 90 hours and after 30 days.
(5) cell injection that redissolves is carried out cell counting and detect, after 90 hours and after 30 days, through automated cell calculating instrument counting, average every bag of amount of viable cell is respectively 8.7 * 10^6,8.6 * 10^6, reach respectively frozen before quantity 87% and 86%.
(6) with taking out the cell injection that redissolves after 90 hours and after 30 days 5 SD rats are carried out intravenous injection respectively, every rat injection volume is 200ul, observes rat through a week and does not produce abnormal response.
Embodiment 2:
(1) according to following recipe configuration 10ml auxiliary material solution:
(2) get human nerve stem cell (source: from the Cord blood that Shanghai Blood Center umbilical cord blood bank gives, extract mescenchymal stem cell, adding growth factor EGF and each 20ng/ml of bFGF induces differentiation to become neural stem cell) 1.0 * 10^7, the centrifugal supernatant of abandoning, it is resuspended to add 10ml auxiliary material solution, through automated cell calculating instrument counting, cell concentration reaches 1.0 * 10^6/ml, makes cell injection, divide to install to 10 frozen pipes of 2ml, every pipe amount of viable cell is 1.0 * 10^6.
(3) put into 4 ℃ 1 hour ,-20 ℃ 1 hour ,-70 ℃ 10 hours, change in the liquid nitrogen and store.
(4) respectively take out 5 solencyte parenteral solutions, 35 ℃ of redissolution respectively at the liquid nitrogen storage after 90 hours and after 30 days.
(5) cell injection that redissolves is carried out cell counting and detect, after 90 hours and after 30 days, through automated cell calculating instrument counting, average every pipe living cells total amount is respectively 9.3 * 10^5,9.21 * 10^5, reach respectively frozen before quantity 93% and 92.1%.
(6) with taking out the cell injection that redissolves after 90 hours and after 30 days 5 SD rats are carried out intravenous injection respectively, every rat injection volume is 200ul, observes rat through a week and does not produce abnormal response.
Embodiment 3:
(1) according to following recipe configuration 100ml auxiliary material solution:
(2) get 1.0 * 10^8 of human mesenchymal stem cell (source: from the Cord blood that Shanghai Blood Center umbilical cord blood bank gives, extract mescenchymal stem cell), the centrifugal supernatant of abandoning, it is resuspended to add 100ml auxiliary material solution, count through the automated cell calculating instrument, cell concentration reaches 1.0 * 10^6/ml, make cell injection, divide to install to 10 frozen blood bags of 50ml, every bag of amount of viable cell is 1.0 * 10^7.
(3) put into 4 ℃ 1 hour ,-20 ℃ 1 hour ,-70 ℃ 10 hours, change in the liquid nitrogen and store.
(4) respectively take out 5 bags of cell injections, 35 ℃ of redissolution respectively at the liquid nitrogen storage after 90 hours and after 30 days.
(5) cell injection that redissolves is carried out cell counting and detect, after 90 hours and after 30 days, through automated cell calculating instrument counting, average every bag of amount of viable cell is respectively 8.55 * 10^6,8.81 * 10^6, reach respectively frozen before quantity 85.5% and 88.1%.
(6) with taking out the cell injection that redissolves after 90 hours and after 30 days 5 SD rats are carried out intravenous injection respectively, every rat injection volume is 300ul, observes rat through a week and does not produce abnormal response.
Embodiment 4:
(1) according to following recipe configuration 10ml auxiliary material solution:
(2) get 1.0 * 10^7 of human mesenchymal stem cell (source: from the Cord blood that Shanghai Blood Center umbilical cord blood bank gives, extract mescenchymal stem cell), the centrifugal supernatant of abandoning, it is resuspended to add 10ml auxiliary material solution, count through the automated cell calculating instrument, cell concentration reaches 1.0 * 10^6/ml, make cell injection, divide to install to 10 frozen pipes of 2ml, every pipe amount of viable cell is 1.0 * 10^6.
(3) put into 4 ℃ 1 hour ,-20 ℃ 1 hour ,-70 ℃ 10 hours, change in the liquid nitrogen and store.
(4) respectively take out 5 solencyte parenteral solutions, 35 ℃ of redissolution respectively at the liquid nitrogen storage after 90 hours and after 30 days.
(5) cell injection that redissolves is carried out cell counting and detect, after 90 hours and after 30 days, through automated cell calculating instrument counting, average every pipe living cells total amount is respectively 9.7 * 10^5,9.44 * 10^5, reach respectively frozen before quantity 97% and 94.4%.
(6) with taking out the cell injection that redissolves after 90 hours and after 30 days 5 SD rats are carried out intravenous injection respectively, every rat injection volume is 300ul, observes rat through a week and does not produce abnormal response.
Claims (9)
1. the frozen protection auxiliary material of a medicinal cell injection contains polyethylene glycol, 1,2-propane diols, albumin and sodium chloride composition.
2. the frozen protection auxiliary material of the cell injection of claim 1, the final concentration of wherein said composition in described cell injection is respectively:
Polyethylene glycol (PEG), 5-100g/L;
1,2-propane diols, 50-400g/L;
Albumin, 10-100g/L;
Sodium chloride, 8.5-9.5g/L.
3. the frozen protection auxiliary material of claim 1 or 2 cell injection, the final concentration of wherein said composition in described cell injection is respectively:
Polyethylene glycol, 50-100g/L,
1, the 2-propane diols, 100-400g/L,
Albumin, 50-100g/L,
Sodium chloride, 8.5-9.5g/L.
4. each the frozen protection auxiliary material of cell injection of claim 1-3, the final concentration of wherein said composition in described cell injection is respectively:
Polyethylene glycol, 80-100g/L,
1, the 2-propane diols, 200-400g/L,
Albumin, 80-100g/L,
Sodium chloride, 9g/L.
5. the frozen protection auxiliary material of each described cell injection of claim 1-4, wherein said albumin is a human serum albumin.
6. the frozen protection auxiliary material of each described cell injection of claim 1-5, wherein said polyethylene glycol is that molecular weight is 400 polyethylene glycol (PEG400).
7. the frozen protection auxiliary material of each described cell injection of claim 1-6, it can also be used as the protectant of biological organization sample glass frozen preservation.
8. the frozen protection auxiliary material of each described cell injection of claim 1-6, it is the frozen protectant of biological organization sample.
Each described frozen protection auxiliary material of claim 1-8 at cell injection the purposes aspect the frozen or biological organization sample glass frozen preservation.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105638642A (en) * | 2016-01-27 | 2016-06-08 | 上海润泉生物技术有限公司 | Immune cell cryopreservation solution and application thereof |
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| US20070087322A1 (en) * | 2005-10-17 | 2007-04-19 | Central Institute For Experimental Animals | Method of preserving early embryos of experimental animals by vitrification |
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| US20070087322A1 (en) * | 2005-10-17 | 2007-04-19 | Central Institute For Experimental Animals | Method of preserving early embryos of experimental animals by vitrification |
| CN101250499A (en) * | 2008-04-11 | 2008-08-27 | 中山大学 | Stem cell nontoxic frozen stock solution |
| WO2010096821A2 (en) * | 2009-02-23 | 2010-08-26 | Cell & Tissue Systems, Inc. | Method for ice-free cryopreservation of tissue |
Non-Patent Citations (1)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105638642A (en) * | 2016-01-27 | 2016-06-08 | 上海润泉生物技术有限公司 | Immune cell cryopreservation solution and application thereof |
| CN105638642B (en) * | 2016-01-27 | 2018-10-19 | 上海润泉生物技术有限公司 | A kind of immunocyte frozen stock solution and its application |
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Application publication date: 20111214 |