SU1089090A1 - N-oxyethylhepta(oxyethylene)-morpholine as cryoprotector - Google Patents

Abstract

H-hydroxyethylhepta (oxyethylene) -morphol in the formula / L (CH2-CH20) 7 - CHj CHjO as a cryoprotectant.

Description

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The invention relates to a chemical compound, specifically to N-hydroxyethylhepta (oxyethylene) -morpholine of the formula (oia-CHto) 7 dn cn, o (used as a cryoprotectant, and may find use in the low-temperature preservation of nuclei containing cells, in particular cell cultures isolated from various organs of animals. The structure closest to the compound of formula (j) is the N-OK seethylmorpholine of the formula, sCHjCHjOH v, which is used to study aminic oxyethylation kinetics l Dimethyl sulfoxide is known (DMSO used as a cryoprotectant in low-temperature preservation of biological objects, in particular, cell cultures. However, DMSO, along with protective properties (preventing or reducing the damaging effects of freezing and thawing on cell cultures during their low-temperature preservation), can have a toxic effect on biological objects In the case of its penetration into the cell, an osmotic gradient arises at the border of the cell in the cell-free medium, which, when transferred to an isoosmotic cell conditions may be the cause of the death of living structures. In order to prevent this damage, it is necessary to remove the cryoprotectant from the cells before transferring them to the physiological osmotic medium. Removing the cryoprotectant from the cells complicates and increases the cost of the whole process of cryopreservation. Thus, DMSO requires cell washing after defrosting, which complicates the process of preservation, since strict asepsis must be observed when washing, and makes it expensive (use of special media, additional staffing). It is not economical because it is used in relatively high concentrations (up to 10%). The formation of a monolayer glue1 0 2 exact cultures, Chyudvergavs with freezing under his protection, occurs only at 4-5 days. It is unstable during storage: decomposes with the formation of sulfur-containing products that have a sharp unpleasant smell (stored for up to 1 month). The purpose of the invention is to simplify and reduce the cost of the cryopreservation process, as well as to increase the degree of protection of biological cultures during low temperature canning. This goal is achieved by using a compound of formula (l) as a cryoprotectant. The compound of formula (1) is obtained by reacting morpholine with ethylene oxide at 90-100 ° C under a nitrogen atmosphere and a pressure of 800-1000 kPa. . 85 g (1 mol) of freshly distilled morpholine is placed in a heated stainless steel reactor equipped with an electric stirrer, a thermocouple, and a device for the discrete feeding of ethylene oxide and a cooling system, and heated to 60-80 ° C. 4-40 g (8 mol) of ethylene oxide are slowly fed in portions of 5-10 ml into the reaction mixture, adjusting the pressure in the range of 800-1000 kPa. The reaction is carried out in an atmosphere of nitrogen, preventing the temperature from exceeding 90-100 ° C. The reaction mixture after feeding in the whole of ethylene oxide is kept for 1-1.5 hours at 70-75 ° C, periodically flushing the reactor with nitrogen. The resulting dark-colored liquid (420 g) is cleaned by repeatedly (3-5 times) processing a 3-4-fold diluted aqueous solution with grade A activated carbon and Ku-2-8c cation-exchange resin. Water and non-reacted initial morpholine are separated from the synthesized compound; they are distilled off in vacuum (0.45 kPa, 27-3lc). The yield of compound (J) 380 g (90%). The product of the synthesis is subjected to further vacuum fractionation (low molecular weight oligomers 0, A-0.5 kPa, 100-150 ° C). Yield of compound of formula (I) 320 g (77%). The structure of the obtained compound is confirmed by elemental analysis, molecular weight and IR spectroscopy. Found,%: C 55.01; H 9.72-; N 3.08. Calculated,%: C 54.66; H 9.33; N 3.19. Molecular weight calculated 436, experimental A25. IR spectra were taken on a Spekord-75 spectrophotometer in CaFj cells. The thickness of the layer is 0.8 mm, the solvent, the residual moisture content in the sample does not exceed 0, 1%. When comparing the IR spectra of the obtained compound and the initial morpholine, it turned out that the strongest discontinuities are observed in the absorption region of the valence vibrations of the amino and oxy groups ( 3600-3200 cm). If in the spectrum of the starting compound the absorption with a maximum of average intensity appears in the absorption band at 3320 cm, referring to the stretching vibrations of the amino group, then in the spectrum of the ethoxylated derivative instead of it there appears a broad complex absorption band at COO -3595 cm, caused by the valence vibrations of the associated hydroxyl groups of the polyoxyethylene chain.j The absence of a band due to the vibrations of the amino group in the IR spectrum of the obtained compound indicates the absence in the target product React union of starting soybeans. The absorption bands of valence and deformation vibrations of CHj-rpynn in the region of 3000-2800 and 1400-1200 cm reflect the nature of the absorption of vibrations of the polyoxyethylene chain. A study of the cryoprotective properties of the compound of formula (I) is carried out as follows. A suspension of kidney cells of a pig embryo in a passage 242 (SPEV) 242 passage and: cryoprotectant, N-hydroxyethylhepta (oxyethylene) morpholine is cooled in a refrigerator up to 4 ° C, after which a suspension of cryoprotector is added to the suspension with gentle mixing in a 1: 1 ratio, prepared on medium 199 with a pH of 7.4. The concentration of cells (final) in a suspension of 8 million in 1 ml. Cells are grown in monolayer on medium 199 with the addition of 10% blood serum of cattle IPO 100 units. penicillin sodium salt and streptomycin calcium chloride complex in 1 ml of nutrient medium. The choice of the optimal cryoprotectant concentration and cell cooling mode is carried out in a multifactorial experiment with different cryoprotectant concentrations from 1.5 to 15% (final concentration) and cooling rates. The best results are obtained when using the CRI protector 4.5-5, 5% concentration. At lower and at higher (15%) concentrations, the formation of a monolayer is significantly (2-3 times) delayed compared to the control (native material). In this case, the monolayer is made up of separate islands, in a number of cases - merging, its design is smoothed. Nutrient replacement is required (1-3 times) or cell transfer. Thus, the deviation of the cryoprotectant concentration from the optimum leads to the fact that the monolayer forging time by 2-3 times exceeds the time of its execution at the optimum concentration. The cell suspension, mixed with a cryoprotectant, is incubated at D С C for 15 minutes, then poured into 2 ml glass ampoules, which, after sealing, are placed in containers. The whole period of equilibration of cells in the cryoprotectant medium is 40 minutes. Suspension freezing is carried out in a two-step program: with speed. minutes to 1 ° C / min up to -196 ° C. Thawing is carried out in a water bath at 41 ° C. After thawing, cell viability is assessed based on the results of intravital trypan blue staining (86%). By morphological features, the SPM cells are retained in a monolayer epithelioid-like shape, have the appearance of an irregular quadrangle. The cytoplasm of cells is homogeneous, a little fine-grained. The nucleus of cells is rounded, less often oval, with 2-5 chips. The mitotic activity of thawed cells does not differ from cells not subjected to: freezing (control). On the first days of growth after thawing in a culture medium on mattresses, the mitotic activity is 33.3 + 1.5%, on the second day 39.0 + 6 , 1% and on the third

J10890906

43, Oil, 5%. The monolayer for the deconservation of the cryoprotective properties of soy and control cells is formed by the dinenes of formula (1) are presented on the third day of growth. The results are examined in the table,

Comparison of the cryoprotective properties of the compound of formula (1) and DMSO

The final concentration of cryoprotectant,%

Washing of thawed cells with double centrifugation

Viability,%

Cell growth in monolayer, days

Storage time The compound of formula (1) as a cryoprotectant provides cryosuction at a concentration of 4.5-5.5%, which is 2 times less than the concentration of DMSO necessary for these purposes. The formation of a monolayer of cells of the SPEV culture after conservation with the compound of formula (1) occurs within 3 days (similar to the control), which reduces the consumption of nutrient media. The compound of formula (I) as a cryoprotector does not require laundering, which simplifies and reduces the cost of preservation, and is stable at xr | 1Nenii (up to 2 years) ..

4.5-5.5

Not Required .8.6

3 2 g Thus, N-hydroxyethylhepta (oxyethylene) -morpholine can be effectively used as a cryoprotectant when freezing cell cultures. The preparative cryoprotector is economical, since it is used in relatively low concentrations, does not have toxicity for cells and, as a result, eliminates its removal from cells by repeated washing, does not reduce the proliferative activity of deconserved cells, which significantly increases productivity.

Claims (1)

N-hydroxyethylhepta (oxyethylene) -morpholine formula about (~ \ - (sn ₂ -sn ₂ o) ₇ - sn ₂ ch ₂ about as a cryoprotectant. one 1089090 · 2