DD140476A1 - METHOD FOR INCREASING THE STABILITY OF TEMPERATURE OF CONSERVATED LIVING CELLS - Google Patents

Abstract

The invention relates to a method for increasing the stability of cells to freeze / thaw damage in cryopreservation. It can be used anywhere where living cells, especially for the purpose of retransmission to similar organisms, should be kept for a long time and kept in stock. The aim of the invention is to significantly increase the membrane stability of the cells and thus their survival outside the organism in comparison to previous possibilities. It is the object of this to develop a suitable method using a stability-increasing active ingredient or a combination of active ingredients. The method according to the invention is characterized in that the preservation solution contains seleno-amino acids, e.g. Seleno-methionine, or a combination of sodium selenite and tocopherol (vitamin E) in a concentration of 1.0 to 10.0 Myg / 100 ml, then the cells to be preserved are suspended therein and incubated for a maximum of 3 h to 37 degrees C. , subsequently freezing in a cryoprotective medium while maintaining a suitable cooling rate and storing in liquid nitrogen. With this method, a high membrane stability of the cells is achieved compared to previously applicable preservation techniques.

Description

Berlin, den'2,10 * 78

Oeinae, Prof »Dr.medehabil · Peter Hackens ellner, Prof # Dr # sc" mecL _e Hans-Alois Ma1rth.es, Dr »med Gert

Jentzsch, Dr "med" vet "habil" Klaus-Dieter Krause, Dr med _{e e} ^x Winfried Jalmbke, ProfV Dr '· as · nat _e Hans-Dieter

Method of increasing the stability of low temperature »

The invention relates to a method for improving the preservation of cells at low Tempez-aturen (iTieftemperaturlcons ervierung) in the sense of an increase in the vital Membraiistabilitä'fc frozen cells with the Sffekt of conservation of function © Ilen όώ / 1 s s ell integrity tr ulrbur The invention can usefully be used everywhere where viable cells are to be stored and stored for a long time, especially in bilateral and blood banks.

The cryogenic preservation of isolated cells is known for almost unlimited time «. For this purpose, v / cells earth in a cryoprotective medium '(physiological medium with the addition, an antifreeze substance such as 2 _ä B "dimethyl sulfoxide or glycerol.) Suspended defined with a certain cooling rate cooled, frozen and deep Üemperaturen (below -80 ⁰ C to -100 ⁰ C ₅ best stored in liquid nitrogen) In this way, z _e Bc red blood cells by applying the ^t? lov- "glycerol-rapid-freezing-techique" (KRUliBIi et al., 1964) or "high-glycerol-freeaing-tecliriiQue" (ECiGGIIiS, 1964)

v © A deep-temperature preservation without the addition of eryoprotectants to the suspension medium has only limited success, since only a small proportion of the cells survive the procedure *

The survival of cryopreserved cells depends largely on the cell type, the cryoprotective additive chosen, and the type of freezing / leu / regime. * The process of cryopreservation, especially of the freeze / thaw process, causes the cells to undergo alterations that are detrimental the stability of the membranes and thus the survival capacity of the cells (PBRSIDSKJ, 1971I MSRyMAT, 1974). So Sebe takes over a subsequent hypοthermon or normotheriaeii storage freeze he preserved 'erythrocyte zjten a progressive Häsiolyse sov. ^7th an increased Ausstroia of potassium ions from the erythrocytes instead (ROWS, 1973 AEBEBXOM, 1974, YALBRI ₈ 1975) o

Overall, it can be stated that the known methods of living cryopreservation cells do not meet all the requirements _e This concerns in particular the lack of stability of erythrocytes during hypothermic storage after previous cryopreservation, which turns out for blood banks to be disadvantageous "

The aim of the invention is to increase the membrane stability of the cells before and / or during tempering door preservation and to make this effect practical, nutable,

It is an object of the invention to develop a process by which the stability of the cells can be increased by suitable additions to the suspending medium.

Erfinöungsgemäß · preservation solution is the addition of the binding target cells to be preserved a suitable Selenver ", preferably a seleno-amino acid or a combination of Hatriumselenit (Na2SeO3) and other additives, preferably vitamins, in a concentration from 1.0 to 10.0 ug For this, the seleno-amino acid, z _e B "seleno-methionine, in a concentration of 2.0 ~ 2.5 μg / 1000 ml, and in the first 3 ° of all the hesiumselenite in one concentration of 1.8 to 2.2 g / 1000 ml and the vitamin, z "B _e Kokopherol (vitamin B), at a concentration of 11 -.! added to 13 ug / 1000 ml * After complete dissolution of these agents, the cells are , which are obtained in a conventional manner, washed and centrifuged, added to the preservative solution and incubated with this up to a maximum of 3 hours at 37 ° C after addition of the cryoprotective medium, which also contains these drugs or drug combinations, and a corresponding incubation for antifreeze distribution takes place while maintaining a suitable cooling rate, the conversion into liquid nitrogen (-196 ⁰ C) for storage over different periods of time «Eei need thawing of the liquid nitrogen removed cells by .Einstellen the preservation container in a Viasserbad with a Temperature of 37 ⁰ O _e

In a review of the structural and functional integrity of the cells can be clearly demonstrated that the method according to the invention significantly increased the proportion of surviving cells compared to similar cell suspensions, which were frozen in the same way, but without addition of the active ingredients to iionservierungslösung, or © the

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markedly degrades part of damaged lines. This increase in the survival of cells in a medium promises "significant economic repercussions everywhere where living cells must be available for the purpose of reuse, particularly for re-transmission to similar organisms" in a viable state, such as, in particular, cell and blood banks "

The invention is explained below using an exemplary embodiment. ·

Whole blood from healthy donors is treated in the ratio 4s1 with AGD-AG stabilizer solutions I-IU (lab, 1), which in the first experimental group seleno-methionine in a consensus of O ₅ , 226 μg / 100 ml, in the was added -second experimental group sodium selenite in a concentration of Oj2 ug / 100 ml and tocopherol in a concentration of 1 _y 2 ug / 100 ml. " The third experimental group serves as a control and contains no active ingredients in the ACD-AG stabilizer solution. For incubation, the .Konservenflaschen be with the preserved blood for 3 hours at 37 ⁰ C kept laugh the end of the incubation period, the preserved blood is centrifuged, the plasma supernatant is discarded and the remaining red blood cells slowly in the ratio 1: 1 with the glycerine preservation solutions I - III (I 2) mixed for 30 min to he G-efri square distribution at 25 ⁰ C held "for cryopreservation, the preserved blood then in centrifuge tubes (0 20 mm, length loo mm) transferred and dipped" directly into liquid nitrogen (-196 ⁰ C) After 24 Thawing is carried out hours or later by placing the centrifuge tubes in a 37 ⁰ G-BaCl 3. After washing the erythrocytes three times, the free hemoglobin content in the supernatant is checked, and the hemolysis rate

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9 0 6 9.1 2

tisa ^ & P ^^

P »in«, - Hemoglobin (mg / 100 ml) in e * in 7 "supernatant. ^ _% ___ «, -________ _-__ χ 100)

Hemoglobin (mg / 100 ml) before freezing

determined »After cryopreservation the hemolysis rate in test group 1 (addition of seleno» methionine) and test group II (addition of sodium selenite and tocopherol oil) is statistically significantly lower than in test group III (control group) _β The results are shown in table 3 «

Composition of the ACD-AG stabilizer solutions used (data in g / l)

Substance 'stabilizer solution

ι; , IIin

Glucose «H2 O 25.0 25.0 substance 25.0 I II III Natriumzitrat'2 H ₂ O 13.2 13.2 13.2 Zionic Acid ⁸ H ^ O 4.8 4.8 4.8 adenine sulfate 0.46 0.46 0.46 guanosine 0.71 0.71 0.71 Sodium elenite 2, Ο · 1Ο " ⁶ - Sokopherol - 1.2 x 10-5 Seleno-methionine 2.26 · 1Ο " ⁶ - Table 2; Composition of used preservatives Solutions (data in g / l) Conservation solution

Glycerol 360.0 360.0 360.0

Sorbitol 30.0 .30.0 30.0

Sodium chloride 60.0 60.0 60.0

Ha tr rams elenit ~ 2, O ^< ? 1O "* ^ ⁾

Eocopherol - 1.2 · 1Ο "* 5

Seleno-methionine 2.26 »'IO" ⁶

Hemolysis rates of cryopreserved erythrocytes using active ingredients (n = 5)

"Experimental Group Hemolysis {%)

I 8:33

II 3.85 ^x)

III 3 _$ 34 ^z )

'Statistically significant compared to experimental group I (p <0 _? 05)

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Claims (3)

Erfindiingsanspruch. 1) Method for increasing the stability of deep "temperature conserved living cells, in particular Blutzelleiiy gek.eianz characterized eichnetj that the cells immediately after their production and to Zn 3 or more hours prior to freezing in a with suitable selenium compounds in a certain concentration j, preferably between 1,0'und 1O "O ug / ml 1OOO, and optionally further to" _s sa.tzstoffen preferably _vitamins? offset 37 ⁰ C warm preservation solution _suspended} then in one cryoprotective medium in defi ~ niertsr manner eingefroreni in f.Iücsigen Sticlcstoff transferred and stored therein to sum demand " 2) Process according to item i) _s characterized,. that of preservation; solution of selenium-methionine, preferably in the. Consentration of 2.0 to 2.5 μg / 1000 ml, added "" will " 3) Procedure according to point 1) _? characterized in that the preservative solution is a combination of sodium ses-serite, preferably in the concentration of 1 _f 8 to 2.2 μg / 1000 ml, and Sokopherol   (Vitamin E), preferably in the concentration of 11 to 13 μg / 1000 ml, is added * /8th