RU2455357C1 - Cell product for auto- and allografting prepared of human umbilical cord, and method for preparing thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: there are offered a cell material and a method for preparing the cell material consisting of mesenchymal cells recovered from human umbilical cord after easy delivery during 38-40 week of pregnancy, and a special transport medium (20-80 vol. %) containing physiologic saline, and a therapeutic solution (20-80 vol. %) specified in the following therapeutic agents: an succinic acid agent, or an anaerobic glycolysis stimulating agent, or a protective agent.

EFFECT: invention enables a faster clinical effect of the cell therapy and a longer shelf life of the cell material; it is applicable for preparing the cell material used for the purpose of auto- and allografting in a wide spectrum of clinical technologies for treating a number of pathological conditions.

8 cl, 3 tbl, 1 ex

Description

The group of inventions relates to medicine and can be used to obtain cellular material used for autologous and allogeneic transplants in a wide range of clinical technologies for the treatment of many pathological conditions.

A modern step in the direction of cell therapy is the study of umbilical cord tissue, human placenta, which are currently considered as one of the optimal sources of stem cells with high progenitor potential.

Cord blood, umbilical cord and placenta tissues are a source of stem cells for transplantation in many diseases, including as severe as blood diseases, hereditary metabolic disorders, pathologies of the central nervous system, etc., and can be used for allogeneic and autologous transplantation.

Stem cells, which are present in large quantities in cord blood, umbilical cord and placenta tissues, have high proliferative activity and are also characterized by the presence of a high titer of pluripotency marker and functional activity of stem cells, the so-called CD34 +.

To date, extensive in vivo experiments continue, confirming high pluripotency, differentiation ability, and a high level of stem cell metabolism derived from placenta, umbilical cord blood, and umbilical cord tissues.

Medical Technologies LLC has developed and protected by a patent (RU 2347579 C1, publ. February 27, 2009) a method for producing a cell culture for treating diseases of the nervous system, including isolating a mesenchymal stem cell culture from the placenta of a newborn for normal delivery for parenteral administration to a neurological patient pathology, cultivating them until a monolayer is formed, and before conducting cell therapy, the monolayer is transferred into a suspension by treatment with a mixture of Versen's solution and trypsin solution, which is then thrice We are washed with physiological saline, then the suspension is precipitated by centrifugation, the cell pellet is resuspended in sterile physiological saline, obtaining allogeneic mesenchymal stem cells of the human umbilical cord in an amount of 0.5-5 million cells in 5 ml of physiological saline.

The same patent describes a technology for producing cell cultures for the treatment of diseases of the nervous system obtained by the above methods and containing from 0.5 to 5 million cells in 5 ml of physiological saline.

A disadvantage of the known methods and cellular materials obtained with their help is that the effect of introducing cellular materials begins to appear only after a month, at best after 2 weeks, as well as by the short shelf life of the cellular material.

The objective of the group of inventions is to improve the consumer properties of cellular material.

The technical result of the group of inventions is to shorten the period before the onset of the effect in the treatment of cellular material while ensuring a long shelf life of cellular material.

The technical result is achieved in that the cellular material, consisting of cells obtained from human placenta after delivery at 38-40 weeks of gestation, and a transport medium containing physiological saline, differs from the known one in that the transport medium also includes a drug in the form a solution selected from the following series depending on the pathology for which the cellular material is intended: a drug based on succinic acid, or a drug that stimulates anae obny glycolysis, or drugs that have a protective effect, in the following ratio,% vol.:

saline solution 20-80,

drug 20-80,

however, the material contains cells in an amount of from 0.1 to 5 million cells in 1 ml of medium for transportation.

Cellular material as a medicine based on succinic acid may include an agent from the series: cytoflavin, mexidol, reamberin.

Cellular material as a medicine stimulating anaerobic glycolysis may include an agent from the series: Actovegin, Cortexin, Cerebrolysin, Solcoseryl.

Cellular material as a protective drug may include an agent from the series: citicoline, levocarnitine, mildronate, berlition, riboflavin, pyridoxine, folic acid, sodium deoxyribonucleate, epoetin-alpha.

The technical result is also achieved by the fact that in the method of obtaining cellular material from human placenta, consisting in the fact that isolated from human placenta after childbirth at 38-40 weeks of gestation and cultured cells are transferred into a suspension and placed in a transport medium containing physiological solution, characterized in that they use a medium for transportation, which also includes a drug in the form of a solution selected from the following series, depending on the pathology, for the treatment of which the adhesive is intended intramural material: the drug on the basis of succinic acid, or a drug that stimulates anaerobic glycolysis, or drugs that have a protective effect, with the following ratio of about.%:

saline solution 20-80,

drug 20-80,

while the finished material contains from 0.1 to 5 million cells in 1 ml of medium for transportation.

In this case, as a medicine based on succinic acid can use a tool from the series: cytoflavin, mexidol, reamberin.

In addition, as a medicine stimulating anaerobic glycolysis, one can use an agent from the series: actovegin, cortexin, cerebrolysin, solcoseryl.

In addition, as a drug that has a protective effect, you can use the drug from the series: citicoline, levocarnitine, mildronate, berlition, riboflavin, pyridoxine, folic acid, sodium deoxyribonucleate, epoetin-alpha.

The essence of the proposed inventions is that the isolated and cultured cellular material is placed in a transport medium containing, along with physiological saline, one of the approved drugs from the above groups, depending on the pathology for the treatment of which the cellular material is intended. Use the finished drug in the form of a solution for injection or for infusion. This provides a significant reduction in the onset of the effect of treatment, as well as a long shelf life of cellular material.

The following is an example implementation of the proposed method for the preparation of cellular material from human placenta and its use. The stage of isolation and cultivation of cellular material is carried out in the same manner as in the known method (RU 2347579).

1. Fence and transportation to the laboratory of biomaterial.

The biomaterial should be taken immediately after delivery at the 38-40th week of gestation in a sterile maternity ward in compliance with all antiseptic rules.

A placenta together with a segment of the human umbilical cord (biomaterial), no later than 10 minutes after ligation and cutting of the umbilical cord, was placed in a sterile airtight container containing Hanks solution with penicillin 100 mg / ml, streptomycin 100 mg / ml, amphotericin 100 mg / ml, which was placed in a cooler bag or ice flask.

The biomaterial was transported to the laboratory sterile and at a temperature of + 2-8 ° C.

2. Isolation and cultivation of stem cells from human placenta (hereinafter referred to as cell material), including the technology of continuous maintenance of cell culture.

All manipulations on the isolation and cultivation of mesenchymal stem cells from human placenta were carried out sterile in the conditions of a specially equipped culture box.

The amnion of the placenta, together with a shallow capture of the chorion, was cut into small pieces (but not less than 1 × 1 cm).

The resulting tissue pieces were washed with Hanks solution. For this, each piece was placed in a separate 50 ml isopropylene tube filled with 40 ml of Hanks solution and shaken vigorously. Then the processed sample was transferred into a new test tube with Hanks solution. Manipulations were performed three times with each piece of tissue.

The washed tissue pieces were crushed and incubated for 2 h at 37 ° C in 0.1% type I collagenase solution in Petri dishes with a diameter of 10 cm at the rate of 1 tissue piece: 1 Petri dish: 8 ml of collagenase solution.

The resulting suspension from each Petri dish was transferred to a separate 15 ml isopropylene tube and precipitated by centrifugation (5 min 300 g).

The resulting precipitates 5 were resuspended each in 10 ml of growth medium: DMEM F12; 10% fetal calf serum; 100 units gentamicin; 2 mM glutamine.

The contents of each tube were transferred to a separate culture vial with an area of 75 cm ² and cultured with a change in growth medium (composition see above) every 3 days until the obtained primary cell culture reached 80% confluency.

When the monolayer reached 80% confluence, the cells were suspended using a solution of 0.25% trypsin with versene (1: 1) and scattered into 75 cm ² culture bottles in a 1: 3 ratio.

An immunochemical analysis of the cellular material was performed for the content of mesenchymal cells (pitophenotype CD45 ^- CD34 ^- CD90 ⁺ CD105 ⁺ ) and testing the percentage of viable cells.

3. Preparation of cellular material for transportation and use.

Selected samples of cell cultures, not older than passage V, were transferred to the suspension using a solution of 0.25% trypsin with versene (1: 1). For this, the growth medium was removed, and the monolayer was washed three times with a solution of 0.25% trypsin with versene, and then incubated in it at 37 ° C at the rate of 3 ml of solution per 75 cm ² culture vial.

The resulting suspension was transferred into a 50 ml isopropylene tube, after which the number of cells was counted using a Goryaev chamber.

Then the suspension was washed with saline. For this, the cells were precipitated by centrifugation (5 min 300 g), and the pellet was resuspended in physiological saline. The manipulation was repeated three times.

The resulting cell pellet was resuspended in a sterile transport medium containing 50% saline with glucose (vol. 0.5%) and 50% actovegin drug, selected taking into account the pathology for the treatment of which the cellular material is intended, namely age-related changes in the skin of the second degrees. Saline and Actovegin were taken in quantities of 1.5 ml.

The preparation of suspensions with other drugs was carried out in a similar manner.

The resulting suspension was transferred sterile into a vacuum blood collection tube (Wackutainer, Beckman Dickenson, USA).

Cellular material for transportation and use was accompanied by a passport of cellular material, including:

- general information: manufacturing time, shelf life, description of appearance, volume of cellular material, storage conditions and labeling of the sample;

- Description of cellular material: cell viability (%), sterility, total cell concentration (cells / ml).

4. Transportation and temporary storage of cellular material.

A. Vacutainer with the finished cellular material was placed in an ice thermos and transported to its destination.

B. Transportation and storage of cellular material was carried out sterile and at a temperature of + 2-8 ° C.

B. If the procedure for applying cellular material did not occur immediately after it arrived at its destination, the vacutainer was either left in a thermos on ice or transferred to a household refrigerator (but in no case to a freezer!).

D. Shelf life of cellular material for medical use was indicated in the accompanying Passport of Cellular Material.

An example of the treatment of patient K., 42 years old.

All medical measures were carried out in the treatment room.

Preparation of cellular material for administration was carried out in compliance with the rules of asepsis and antiseptics.

Immediately prior to administration, the contents of the tube were shaken several times to distribute the cellular material evenly and the cellular material was transferred to an injection syringe.

Before cell therapy, skin cleansing was carried out using solutions containing surface-active detergents. Skin disinfection was performed at the injection site with a cotton pad dipped in a 0.05% solution of chlorhexidine bigluconate. After this, the antiseptic solution was washed off with sterile physiological saline, the skin and wound were dried with a sterile napkin, and the chipping zone was covered with sterile material.

The introduction of cellular material was carried out using 5.0 ml syringes with special needles for intradermal injection.

Cellular material was injected using multiple intradermal injections (by mesotherapy technique) into the skin to the depth of the papillary dermis (2 mm), while the volume of the injected drug should be 0.02-0.05 ml per 1 injection point. During the introduction, the micropapule technique was used, since using this technique the necessary depot of the drug is created in the tissues.

The technique of micropapules. Cellular material was injected with separate injections to form papules. Injections were performed over the entire surface of the cleaved zone at a distance of 8-10 mm from each other. 1-2 injections per 1 cm ^{2 of} skin. The syringe and needle were placed tangentially to the skin, the needle section was directed upwards. Pressure on the piston was carried out with the thumb of the working hand.

Duration of the course: a treatment course using cellular material is designed for 2 procedures with an interval of 1 month. The amount of cellular material introduced per 1 session ranged from 1.0 × 10 ⁶ cells to 5.0 × 10 ⁶ cells and depended on the severity of the problem.

In order to maintain the achieved effect, it is advisable to conduct another procedure 6 months after the main course.

On the second day after the first procedure, both in the opinion of the patient and according to the instrumental examination, the clinical effect began to appear.

Table 1 shows examples of compositions of cellular materials according to the proposed invention, intended for the treatment of various pathologies.

Table 1 Type of pathology Medicine Cell volume, million The volume of the transport medium, ml The composition of the transport medium,% lek. facilities 1. Age-related changes of the 2nd degree Actovegin 3 3 fifty% 2. Age-related changes of the 3rd degree Solcoseryl 5 3 80% 3. Age-related changes, eyelids Cerebrolysin one 2 twenty% 4. Alopecia Reamberin 2 5 fifty% 5. Focal allopecia Reamberin one 2 twenty% 6. Scars Mexidol 0.5 one fifty% 7. Trophic ulcer, up to 1% of the body Cytoflavin 3 5 fifty% 8. Burn wound, up to 1% of the body Riboflavin 3 3 fifty% 9. Burn wound, up to 3% of the body Mildronate 5 5 80% 10. Pressure ulcers, up to 1% of the body Berlition 3 four fifty%

The effectiveness of the application of the proposed cellular material is based on physiological stimulation of neoangiogenesis - the growth of new young and intact vessels, as well as activation beyond the physiological level of the recovery and regenerative potential of the patient’s own structures (cells) of the patient’s body. The effect begins to manifest itself after 2-4 weeks and then gradually increases.

The use of cellular material in a transport medium containing physiological saline with glucose (vol. 0.1% -20%) and one of the permitted drugs from the above groups, depending on the pathology, can significantly reduce the time before the onset of the effect (see table 2) .

table 2 Type of drug Effect onset Cellular material in a drug environment Cellular Material in Saline Actovegin (age-related changes of the 2nd degree) From the 2nd day From the 14th day Solcoseryl (age-related changes of the 2nd degree) From the 2nd day From the 14th day Cerebrolysin (age-related changes of the 2nd degree) From the 2nd day From the 14th day Reamberin (trophic ulcer up to 1% of the body) From the 4th day From the 16th day Mexidol (trophic ulcer up to 1% of the body) From the 4th day From the 16th day Cytoflavin (trophic ulcer up to 1% of the body) From the 4th day From the 16th day Riboflavin (burn wound, up to 1% of the body) From the 2nd day From the 6th day Berlition (burn wound, up to 1% of the body) From the 2nd day From the 6th day Mildronate (burn wound, up to 1% of the body) From the 2nd day From the 6th day

The proposed method and cellular material provide an increase in the shelf life of cellular material (a standard shelf life of 6 hours in a transport medium is physiological saline). A similar result was obtained for other drugs of the indicated groups: cortexin, citicoline, levocarnitine, pyridoxine, folic acid, sodium deoxyribonucleate, epoetin-alpha.

The results of testing the survival of cellular material using different transport media (step accuracy of 5%, norm of at least 80%) are shown in table 3.

Table 3 Drug groups The number of viable cells,% 6 h 14 h 24 h 36 h 48 h Saline 85% twenty% 0% 0% 0% Based on succinic acid: - reamberin; 95% 90% 85% 75% 65% - Mexidol; 95% 90% 84% 74% 65% - cytoflavin. 96% 91% 87% 76% 68% Anaerobic Glycolysis Stimulating: - Actovegin; 98% 95% 90% 85% 70% - cerebrolysin; 97% 94% 90% 83% 68% - solcoseryl. 98% 94% 90% 83% 68% Providing a protective effect: - riboflavin; 95% 95% 90% 80% 65% - mildronate; 95% 95% 91% 80% 67% - berlition. 96% 95% 91% 82% 68%

An increase in the percentage of cell survival was observed for other drugs of the indicated groups: cortexin, citicoline, levocarnitine, pyridoxine, folic acid, sodium deoxyribonucleate, epoetin-alpha.

Claims (8)

1. A cellular product for autologous and allogeneic transplantations, consisting of mesenchymal stem cells obtained from human placenta after childbirth at 38-40 weeks of gestation, and a transport medium containing physiological saline, characterized in that the transport medium also includes a drug in as a solution selected from the following series: succinic acid drug, or anaerobic glycolysis stimulating drug, or leaking drug active effect, in the following ratio, vol.%:
medicine 20-80 saline the rest is up to 100,
wherein the product contains cells in an amount of from 0.1 to 5 million cells in 1 ml of transport medium. 2. The cell product according to claim 1, characterized in that as a medicine based on succinic acid includes a means from the series: cytoflavin, mexidol, reamberin. 3. The cell product according to claim 1, characterized in that as a drug stimulating anaerobic glycolysis, it includes an agent from the series: Actovegin, Cortexin, Cerebrolysin, Solcoseryl. 4. The cell product according to claim 1, characterized in that as a medicinal product having a protective effect, it includes an agent from the series: citicoline, levocarnitine, mildronate, berlition, riboflavin, pyridoxine, folic acid, sodium deoxyribonucleate, epoetin-alpha. 5. A method of obtaining a cell product for autologous and allogeneic transplantation from human placenta, which consists in the fact that isolated from human placenta after childbirth at 38-40 weeks of gestation and cultured mesenchymal stem cells are transferred into a suspension and placed in a transport medium containing physiological saline , characterized in that they use a medium for transportation, which also includes a drug in the form of a solution selected from the following series: drug based on amber to acid, or a drug that stimulates anaerobic glycolysis, or a drug that has a protective effect, in the following ratio, vol.%:
medicine 20-80, saline the rest is up to 100,
while the finished product contains from 0.1 to 5 million cells in 1 ml of medium for transportation. 6. The method according to claim 5, characterized in that as a medicinal product based on succinic acid, a means from the series: cytoflavin, mexidol, reamberin is used. 7. The method according to claim 5, characterized in that as a drug stimulating anaerobic glycolysis, use is made of a series of Actovegin, Cortexin, Cerebrolysin, Solcoseryl. 8. The method according to claim 5, characterized in that as a medicinal product having a protective effect, use is made of a series of citicoline, levocarnitine, mildronate, berlition, riboflavin, pyridoxine, folic acid, sodium deoxyribonucleate, epoetin-alpha.