ANTI-LESION STORAGE METHOD AND STORAGE SYSTEM FOR CELLS, TISSUES OR ORGANS

Abstract

The disclosure discloses an anti-lesion storage method and storage system for cells, tissues and organs, and relates to the technical field of storage of cells, tissues or organs. The method includes: adjusting a partial pressure of carbon dioxide in a suspension medium containing cells, tissues or organs to be stored to 30-50 mmHg. The inventors found that the regulation of the partial pressure of carbon dioxide in the suspension medium is advantageous for improving anti-lesions of cells, tissues or organs. In particular, the regulation of the partial pressure of carbon dioxide in the suspension medium can prolong the in vivo survival of platelets after transfusion of a platelet concentrate and protect the hemostatic function of platelets whiling effectively alleviating PSLs.

Claims

1. An anti-lesion storage method for cells, tissues or organs, the method comprising: adjusting a partial pressure of carbon dioxide in a suspension medium containing cells, tissues or organs to be stored to 30-50 mmHg; wherein preferably, the cells are selected from platelets; and more preferably, the cells are selected from platelet concentrate

2. The method according to claim 1, wherein the method comprises: placing the platelet concentrate to be stored in a gas permeable container in a three-gas storage apparatus, and controlling a volume fraction of carbon dioxide in the three-gas storage apparatus to be 1-8%; and preferably, the volume fraction of carbon dioxide in the three-gas storage apparatus is controlled to be 2-5%.

3. The method according to claim 2, wherein the platelet concentrate to be stored is placed in the gas permeable container in the three-gas storage apparatus, and a volume fraction of oxygen in the three-gas storage apparatus is controlled to be 10-20%; and preferably, the volume fraction of nitrogen in the three-gas storage apparatus is controlled to be 70-90%.

4. The method according to claim 2, wherein a storage temperature in the three-gas storage apparatus is controlled to be 2-24° C., and a storage condition comprises: standing storing when the storage temperature is 2-17° C., or keeping continuous shaking when the storage temperature is 18-24° C.

5. The method according to claim 1, wherein the method comprises: placing the platelet concentrate to be stored in an air-tight container, and filling the air-tight container with one of, any combination of two of, or a mixture of all of carbon dioxide, oxygen and nitrogen, with a volume fraction of carbon dioxide being 1-8%, a volume fraction of oxygen being 10-20%, and a volume fraction of nitrogen being 70-90%; and preferably, placing the air-tight container containing the platelet concentrate to be stored in the storage apparatus, controlling a storage temperature in the storage apparatus to be 2-24° C., wherein a storage condition comprises: standing storing when the storage temperature is 2-17° C., and shaking storing when the storage temperature is 18-24° C.

6. The method according to claim 1, wherein the method comprises: placing the platelet concentrate to be stored in a container equipped with a one-way gas valve or in a container made of a one-way gas permeable functional material; and preferably, placing the container having one-way gas permeable function and containing the platelet concentrate to be stored in the storage apparatus, controlling a storage temperature in the storage apparatus to be 2-24° C., and allowing the platelet concentrate to keep standing when the storage temperature is 2-17° C., or keeping shaking when the storage temperature is 18-24° C.

7. The method according to claim 6, wherein a substance capable of producing carbon dioxide by a chemical reaction is added to the suspension medium for the platelet concentrate; preferably, the substance capable of producing carbon dioxide by a chemical reaction is selected from at least one of carbonic acid, a carbonate, and an enzyme; and preferably, the carbonate is selected from calcium carbonate or sodium bicarbonate, and the enzyme is selected from hexokinase, phosphofructokinase-1, 3-phosphoglyceraldehyde dehydrogenase, phosphoglyceraldehyde kinase, pyruvate kinase, pyruvate dehydrogenase complex, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase complex, succinyl-CoA synthase, succinate dehydrogenase, malate dehydrogenase, fatty acyl-CoA synthase, L-3-hydroxy-4-trimethylammonium butyric acid, carnitine acyltransferase I, carnitine acyltransferase II, β-ketoacyl-CoA thiolase, fatty acyl-CoA dehydrogenase, enoyl-CoA hydratase, or β-hydroketothiolase of β-hydroxy fatty acyl-CoA dehydrogenase.

8. The method according to claim 1, wherein a platelet collection device containing the platelets to be stored is pre-filled with any combination of two of, or a mixture of all of carbon dioxide, oxygen and nitrogen, with a volume fraction of carbon dioxide being 1-8%, a volume fraction of oxygen being 10-20%, and a volume fraction of nitrogen being 70-90%; and preferably, the partial pressure of carbon dioxide in the suspension medium for the platelet concentrate is controlled to 30-50 mmHg during collection of the platelets to be stored.

9. The method according to claim 8, wherein the collection device for the platelets to be stored is pre-filled with a substance capable of producing carbon dioxide by a chemical reaction; preferably, the substance capable of producing carbon dioxide by a chemical reaction is selected from at least one of carbonic acid, a carbonate or an enzyme; preferably, the carbonate is selected from calcium carbonate or sodium bicarbonate, and the enzyme is selected from hexokinase, phosphofructokinase-1, 3-phosphoglyceraldehyde dehydrogenase, phosphoglyceraldehyde kinase, pyruvate kinase, pyruvate dehydrogenase complex, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase complex, succinyl-CoA synthase, succinate dehydrogenase, malate dehydrogenase, fatty acyl-CoA synthase, L-3-hydroxy-4-trimethylammonium butyric acid, carnitine acyltransferase I, carnitine acyltransferase II, β-ketoacyl-CoA thiolase, fatty acyl-CoA dehydrogenase, enoyl-CoA hydratase, or β-ketothiolase of β-hydroxy fatty acyl-CoA dehydrogenase; and preferably, an organism of the platelet concentrate to be stored is selected from human, monkey, sheep, rabbit, mouse, swine, dog, cat, horse, or cow.

10. A storage system for cells, tissues or organs, wherein the system can control a partial pressure of carbon dioxide in a suspension medium containing the cells, tissues or organs to be stored to be 30-50 mmHg, and the system comprises a one-way gas permeable container for storing the cells, tissues or organs, a carbon dioxide-releasing substance, and a storage apparatus; the carbon dioxide-releasing substance is selected from at least one of carbonic acid, a carbonate, or an enzyme; preferably, the cells are selected from platelets; and preferably, the cells are selected from platelet concentrate.

11. The method according to claim 3, wherein a storage temperature in the three-gas storage apparatus is controlled to be 2-24° C., and a storage condition comprises: standing storing when the storage temperature is 2-17° C., or keeping continuous shaking when the storage temperature is 18-24° C.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to illustrate the technical solutions of the examples of the present disclosure more clearly, the accompanying drawings used for the examples will be briefly introduced. It should be appreciated that the following drawings show some examples of the present disclosure only, and thus should not be construed as a limitation of the scope. For those of ordinary skill in the art, other related drawings can also be obtained according to these drawings without any creative effort.

[0049] FIG. 1A shows statistical data of CD62P expression of the platelets stored in Examples and Comparative Examples;

[0050] FIG. 1B shows statistical data of PS eversion of the platelets stored in Examples and Comparative Examples;

[0051] FIG. 1C shows statistical data of CD42b shedding of the platelets stored in Examples and Comparative Examples;

[0052] FIG. 2A shows statistical data of glucose consumption of the platelets stored in Examples and Comparative Examples;

[0053] FIG. 2B shows statistical data of lactic acid production of the platelets stored in Examples and Comparative Examples;

[0054] FIG. 2C shows statistical data of ATP concentration of the platelets stored in Examples and Comparative Examples;

[0055] FIG. 3A shows statistical data of aggregation function of the platelets stored in Example 1 and Comparative Example 4 using AA as an inducer;

[0056] FIG. 3B shows statistical data of aggregation function of platelets stored in Example 1 and Comparative Example 4 using ADP as an inducer;

[0057] FIG. 4 shows statistical data of MA values of the platelets stored in Example 1 and Comparative Example 4; and

[0058] FIG. 5 shows statistical data of post-transfusion survival of platelets stored in Example 1 and Comparative Example 4.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0059] To make the objects, technical solutions, and advantages of the examples of the present disclosure more clear, the technical solutions in the examples of the present disclosure will be described clearly and completely below. If the specific conditions are not indicated in the examples, the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturers. The used reagents or instruments without indication of the manufacturer are conventional products that can be purchased from the market.

[0060] The features and performances of the present disclosure will be described in further detail below in conjunction with the examples.

[0061] Main reagents and materials in this example include: [0062] Anti-human CD62P-FITC antibody; Annexin V-FITC apoptosis detection kit available from BD Company, USA; Anti-human CD42b-PE, Anti-human CD41-FITC and Anti-mouse CD41-APC available from Biolegend Company, USA; arachidonic acid (AA) detection kit for platelet aggregation function and adenosine diphosphate (ADP) detection kit for platelet aggregation function available from Sysmex Corporation, Japan; ATP detection kit available from Shanghai Beyotime Biotechnology Co., Ltd.; Amicus Separator, a platelet separator, and attached platelet storage bag (Amicus, USA), 3131 three-gas incubator (Thermo, USA), SYNERGY microplate reader (Bio-Tex, USA), FACS Canto flow cytometer (BD, USA), GEM3500 blood gas analyzer (IL, USA), CS-2400 automated blood coagulation analyzer (Sysmex, Japan), 5000 thromboelastography analyzer (Haemoscope, USA), and non-obese diabetes server combined immune-deficiency (NOD SCID) mice available from Cyagen Biosciences Inc.

Example 1

[0063] This example provides a platelet storage method including: controlling a partial pressure of carbon dioxide in a suspension medium for a platelet concentrate to be stored to be 40 mmHg.

[0064] Specifically, the platelet concentrate to be stored was first prepared as follows. A healthy adult who had not taken any medicament within the past 10 days was recruited as a volunteer and allowed to sign the informed consent form. General examinations were conducted on the blood donor in accordance with the requirement of health examinations for blood donors. After passing the examinations, the volunteer was guided for the blood collection, and 20 U of fresh platelets were directly collected from the volunteer using a blood cell separator. The collected platelets were stored under shaking at 22±2° C. for later use.

[0065] The above platelets were charged in a gas permeable platelet storage bag, then placed in a three-gas incubator, and stored under shaking at 22±2° C.

[0066] The gas composition in the three-gas incubator was controlled to be 3.5% CO2, 17%02, and 80% N2.

Example 2

[0067] The present example provides a platelet storage method including: charging a platelet concentrate to be stored in an air-tight container, filling the container with a mixed gas including 3.5% carbon dioxide, 17% oxygen, and 80% nitrogen, all in volume fractions, then placing the container in a storage apparatus, and storing it under shaking at 22±2° C. The remaining steps were the same as in Example 1.

Example 3

[0068] The present example provides a platelet storage method including: charging a platelet concentrate to be stored in a container equipped with a one-way gas valve that only allows entry of gas outside, and charging sodium bicarbonate in the container in advance, then placing the container in a storage apparatus, and storing it under shaking at 22±2° C. The remaining steps were the same as in Example 1.

Comparative Example 1

[0069] The only difference from Example 1 is that the partial pressure of carbon dioxide in the suspension medium for the platelet concentrate to be stored was controlled to be 5 mmHg.

Comparative Example 2

[0070] The only difference from Example 1 is that the partial pressure of carbon dioxide in the suspension medium for the platelet concentrate to be stored was controlled to be 30 mmHg.

Comparative Example 3

[0071] The only difference from Example 1 is that the partial pressure of carbon dioxide of the suspension medium of the platelet concentrate to be stored was controlled to be 50 mmHg.

Comparative Example 4

[0072] The only difference from Example 1 is that the platelet concentrate to be stored was charged in a traditional gas permeable platelet storage bag, then placed in a shaking storage apparatus for platelets, and stored under shaking at 22±2° C.

Experimental Example 1

[0073] The platelets of the above-mentioned Examples and Comparative Examples were analyzed for CD62P expression, PS eversion, and D42b shedding. Experiment grouping and respective treatments were performed as follows. The platelets to be used were equally divided into 7 groups, and these groups were stored according to the methods of Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4, respectively. Then, an appropriate amount of each of samples to be tested was taken on the 3rd, 5th, 7th and 8th days of storage, and marking for CD62P, PS, and CD42b was conducted with reference to the instructions of Anti-CD62P-FITC antibody, Annexin V-FITC apoptosis detection kit, and Anti-CD42b-PE antibody, respectively, followed by flow cytometry.

[0074] FIG. 1A, FIG. 1B, and FIG. 1C show the statistical analysis of CD62P expression, PS eversion, and CD42b shedding in the respective groups on the 3rd, 5th, 7th and 8th days of storage. It can be found that, from the results of Examples 1 and 4 comparative examples, with the increase of CO2 partial pressure in the suspension medium for the platelet concentrate, both the CD62P-positive platelet ratio representing CD62P expression and Annexin V-positive platelet ratio representing PS eversion first decreased and then increased, while the mean fluorescence intensity of PE representing CD42b shedding first increased and then decreased. In the method of Example 1, the CD62P-positive platelet ratio and Annexin V-positive platelet ratio were the lowest, and the mean fluorescence intensity was the highest. The CD62P-positive platelet ratio and Annexin V-positive platelet ratio in Example 1, Example 2, and Example 3 decreased, and the mean fluorescence intensity of PE increased, compared with Comparative Example 4, for the same storage duration. The CD62P-positive platelet ratio, Annexin V-positive platelet ratio, and the mean fluorescence intensity of PE in Example 1, Example 2, and Example 3 on the 7th day of storage were not statistically different from those of Comparative Example 4 on the 5th day of storage. CD62P expression, PS eversion, and CD42b shedding were found when PSLs occurred. Therefore, the above results indicate that the partial pressure of CO2 in the suspension medium for the platelet concentrate to be stored is associated with the occurrence of PSLs. The methods used in Example 1, Example 2, and Example 3 all increase the CO2 partial pressure in the suspension medium for the platelet concentrate to be stored to 40 mmHg, thereby delaying the occurrence of PSLs. In addition, it is also suggested that the methods used in Example 1, Example 2, and Example 3 can extend the in vitro storage life of platelets from 5 days in a conventional method to 7 days while improving the in vitro storage effect of platelets.

Experimental Example 2

[0075] The blood gas analysis was performed on the platelet groups of the above-mentioned Examples and Comparative Examples. The only difference from Experimental Example 1 was that the blood gas analysis was performed with reference to the instructions of the blood gas analyzer, and the ATP concentration analysis was performed with reference to the instructions of the ATP detection kit.

[0076] FIG. 2A, FIG. 2B, and FIG. 2C show the statistical analysis of glucose consumption, lactic acid production, and ATP concentration of the respective groups on the 3rd, 5th, 7th and 8th days of storage. It can be found that, from the results of Examples 1 and 4 comparative examples, with the increase of CO2 partial pressure in the suspension medium for the platelet concentrate, both glucose consumption and lactic acid production first decreased and then increased, and ATP concentration first increased and then decreased. In the method of Example 1, the glucose consumption and lactic acid production were the lowest, and ATP concentration was the highest. The glucose consumption and lactic acid production in Example 1, Example 2, and Example 3 all decreased, and the ATP concentration was increased, compared with Comparative Example 4, for the same storage duration. The glucose consumption, lactic acid production and ATP concentration in Example 1, Example 2, and Example 3 on the 7th day of storage were not statistically different from those of Comparative Example 4 on the 5th day of storage. ATP production was also reduced when PSLs occurred, which was accompanied by rapid glucose consumption and massive accumulation of lactic acid. Therefore, the above results further indicate that the partial pressure of CO2 in the suspension medium for the platelet concentrate to be stored is associated with the occurrence of PSLs. The methods used in Example 1, Example 2, and Example 3 all increase the CO2 partial pressure in the suspension medium for the platelet concentrate to be stored to 40 mmHg, thereby delaying the occurrence of PSLs. In addition, it is further suggested that the methods used in Example 1, Example 2, and Example 3 can extend the in vitro storage life of platelets from 5 days in a conventional method to 7 days while improving the in vitro storage effect of platelets.

Experimental Example 3

[0077] The platelets stored in Example 1 and Comparative Example 4 above were detected for their aggregation function. An appropriate amount of each of samples to be tested was taken on the 3rd, 5th and 7th days of storage. The platelet aggregation function thereof was detected using an automated blood coagulation analyzer in reference to the instructions of AA detection kit for platelet aggregation function (turbidimetric method) and ADP detection kit for platelet aggregation function (turbidimetric method).

[0078] FIG. 3A and FIG. 3B show the statistical analysis of the two platelet aggregation tests using AA or ADP as inducers, respectively. The results showed that AA-induced or ADP-induced platelets stored in both Example 1 and Comparative Example 4 had reduced aggregation rates over time. The platelets stored in Example 1 had increased aggregation rate compared with Comparative Example 4, for the same storage duration. In addition, the aggregation rate of the platelets stored in Example 1 on the 7th day of storage was not statistically different from that of the platelets stored in Comparative Example 4 on the 5th day of storage. The aggregation rate of platelets is proportional to the hemostatic function of platelets. Therefore, these results indicate that increasing the CO2 partial pressure in the suspension medium for in vitro storage of the platelet concentrate to 40 mmHg can protect the hemostatic function of platelets, further suggesting that increasing the CO2 partial pressure in the suspension medium for in vitro storage of the platelet concentrate to 40 mmHg not only improves the in vitro storage effect of platelets, but also extends the in vitro storage life of platelets from 5 days in a conventional method to 7 days.

Experimental Example 4

[0079] MA values of the platelets stored in Example 1 and Comparative Example 1 above were detected. A healthy adult who had not taken any medicament within the past 10 days was recruited as a volunteer. After disinfection, 3 mL of peripheral blood was collected from median cubital vein and charged in a test tube containing 3.8% sodium citrate anticoagulant, centrifuged at 100 g for 10 minutes to separate platelet-rich plasma and cells in the lower layer, and then centrifuged at 2000 g for 10 minutes to separate the platelets and plasma in the platelet-rich plasma. Then, the obtained plasma was mixed with the cells in the lower layer obtained before to obtain platelet-deficient human peripheral blood. Subsequently, the human platelets stored in Example 1 and Comparative Example 1 were each added to the platelet-deficient human peripheral blood to give a final concentration of 200×109/L. After mixing thoroughly, the MA value was detected in reference to the instructions of the kit.

[0080] FIG. 4 shows the statistical analysis of the MA values of the two groups on the 5th and 7th day. The results found that the MA value of Example 1 was higher than that of Comparative Example 1, for the same storage duration. In addition, there was no statistical difference between Example 1 on the 7th day of storage and Comparative Example 1 on the 5th day of storage. The MA value is one of the classic indicators that comprehensively reflect hemostatic function of platelets, and is proportional to hemostatic function of platelets. This result further confirms that increasing the CO2 partial pressure in the suspension medium for in vitro storage of the platelet concentrate to 40 mmHg can protect the hemostatic function of platelets, further suggesting that increasing the CO2 partial pressure in the suspension medium for in vitro storage of the platelet concentrate to 40 mmHg can extend the in vitro storage life of platelets from 5 days in a conventional method to 7 days.

Experimental Example 5

[0081] In this experimental example, the platelets stored in Example 1 and Comparative Example 1 above were subjected to analysis and statistics of the post-transfusion survival. An appropriate amount of each of samples to be tested were taken on the 3rd, 5th and 7th days of storage, and then transfused into mice through the tail vein. Then, an appropriate amount of peripheral blood was collected from the tip of the tail 12 hours after transfusion. The peripheral blood after lysis of red blood cells were co-labeled with Anti-human CD41-FITC and Anti-mouse CD41-APC. The ratio of FITC+/APC+ was analyzed by flow cytometry.

[0082] FIG. 5 shows the statistical data of post-transfusion survival of the two groups of platelets. The results showed that, the ratio of FITC+/APC+ of the platelets stored in Example 1 and Comparative Example 1 both decreased after transfusion over time after transfusion, and the ratios of FITC+/APC+ of Example 1 after transfusion were all higher than those of Comparative Example 1, for the same storage duration. In addition, there was no statistical difference between Example 1 on the 7th day of storage and Comparative Example 1 on the 5th day of storage. This result shows that increasing the CO2 partial pressure in the suspension medium for in vitro storage of the platelet concentrate to 40 mmHg can extend the post-transfusion survival of platelet, and also indicates that increasing the CO2 partial pressure in the suspension medium for the platelet concentrate to 40 mmHg can extend the in vitro storage life of platelets from 5 days in a conventional method to 7 days.

[0083] It is well known to those skilled in the art that PSLs would occur in platelets during in vitro storage. Specific manifestation of PSLs includes CD62P expression, CD42b shedding, PS eversion, glucose consumption, lactic acid accumulation, and reduction in ATP production occurred in platelets, along with impaired hemostatic function, shortened post-transfusion survival, and so on. A series of studies made by the inventors found that increasing the partial pressure of CO2 in the storage medium to 40 mmHg can effectively alleviate activation, apoptosis, and CD42b shedding of platelets stored. Furthermore, after the partial pressure of CO2 in the storage medium to 40 mmHg, the hemostatic function was also greatly improved, and the post-transfusion survival was significantly prolonged.

[0084] In conclusion, the technical solution disclosed in the present disclosure is a new strategy for enhancing storage effect for platelets and prolonging their storage life by improving the in vitro storage conditions for platelets, and is of great significance for the storage of platelets.

[0085] The above descriptions are only preferred examples of the present disclosure, and are not intended to limit the present disclosure. For those skilled in the art, various modifications and changes can be made to the present disclosure. Any modification, equivalent replacement, improvement, and the like made within the spirit and principle of the present disclosure shall be included within the protective scope of the present disclosure.