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WO2012154016A1 - Procédé de production de lyophilisats à partir d'hépatocytes humains - Google Patents

Procédé de production de lyophilisats à partir d'hépatocytes humains Download PDF

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Publication number
WO2012154016A1
WO2012154016A1 PCT/KZ2011/000009 KZ2011000009W WO2012154016A1 WO 2012154016 A1 WO2012154016 A1 WO 2012154016A1 KZ 2011000009 W KZ2011000009 W KZ 2011000009W WO 2012154016 A1 WO2012154016 A1 WO 2012154016A1
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WIPO (PCT)
Prior art keywords
hepatocytes
cells
fetal
liver
patients
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/KZ2011/000009
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English (en)
Russian (ru)
Inventor
Абай Кабатаевич БАЙГЕНЖИН
Болат Амангельдиевич КАЮПОВ
Самат Сагатович САПАРБАЕВ
Манарбек Бапович АСКАРОВ
Жаксылык Акмурзаевич ДОСКАЛИЕВ
Айжан Абдисадиковна АХАЕВА
Айгерим Хайруллаевна ЖАКУПОВА
Куаныш Дюсембекович СЕМБАЕВ
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
AKCIONERNOE OBSHECTVO "NACIONALNI NAUCHNI MEDICINCKI CENTR"
Original Assignee
AKCIONERNOE OBSHECTVO "NACIONALNI NAUCHNI MEDICINCKI CENTR"
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by AKCIONERNOE OBSHECTVO "NACIONALNI NAUCHNI MEDICINCKI CENTR" filed Critical AKCIONERNOE OBSHECTVO "NACIONALNI NAUCHNI MEDICINCKI CENTR"
Priority to PCT/KZ2011/000009 priority Critical patent/WO2012154016A1/fr
Publication of WO2012154016A1 publication Critical patent/WO2012154016A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/407—Liver; Hepatocytes

Definitions

  • a method of obtaining a lyophilisate from human hepatocytes is a method of obtaining a lyophilisate from human hepatocytes.
  • fetal material When testing fetal material, a standardized certification scheme for fetal material was used, which corresponds to international standards for infection of tissues used in the treatment of fetal tissues and cells. Fetal material was tested by PCR for the following pathogens: chlamydia trachomatis, chlamydia pnevmonica, ureaplasmas urelyticum, ureaplasmas parvum, mycoplasma hominis, mycoplasma genitalium, neiseria gonorrginalis, 2 gonorgalis virus, gororinal gerorinova, gonorginalis 2 (herpevirus), HSV 16 (papillomavirus), HPV 18 (papillomavirus), HSV 6 (herpevirus), Candida albicans, treponema pallidum, toxoplasma gondii, mycobacterium tuberculosis, hepatitis A virus, hepatitis B virus, hepatit
  • the object of the study was the fetal liver. Material was taken immediately after abortion, according to the original methodology developed by the scientific group, followed by processing and homogenization using a device to separate cells from the stroma.
  • a donor card was filled out for each fetus.
  • the terms of the fetus ranged from 18 to 22 weeks of gestation (Table 1).
  • the fruits took into account gender, body weight, embryo growth, the characteristics of which are shown in table 2.
  • hepatic duct ductus hepatikus
  • portal vein vena porta
  • posterior vena cava veins v. cava posterior
  • arteries of the liver a. hepatica
  • common bile duct ductus choledohus
  • duct of the gallbladder ductus cysticus
  • ligaments of the liver coronary (lig. coronarium hepatis), triangular (lig. triangulare), sickle-shaped (lig.
  • hepatic-renal (li g. hepatorenale), hepatic-gastric (lig. hepatogastricum), hepatoduodenal (lig. hepatoduodenale).
  • liver will be removed and with the removal of the gallbladder (vesica fellea).
  • gallbladder vesica fellea
  • the liver is placed in a sterile Petri dish, for further mechanical processing on a homogenizer.
  • the above homogeneous suspension of dissociated cells with a Pasteur pipette was layered on the density gradient in the ratio of the volume of the gradient and the shared suspension 1: 2 - 1: 5.
  • the tubes are centrifuged at room temperature for 20 minutes with a centrifugal acceleration of 400g ("1550 rpm).
  • the supernatant containing leukocytes is removed with a Pasteur pipette, the sedimentary fraction, which is a suspension of fetal cells, is washed with an isotonic solution by centrifugation for 5 minutes with a centrifugal acceleration of 200 g (“775 rpm).
  • Cell viability is determined using a vital dye - trypan blue: a drop of the resulting suspension was stained with a 0.1% trypan blue solution, placed in a hemocytometer and examined under a microscope. Living, normally functioning cells are not stained with this dye ( Figure 2).
  • distilled water in a ratio of 1: 3 was added to the resulting homogenate for 30 seconds in order to remove “passenger” cells (lysis of the remaining blood cells and destruction of mechanically damaged embryonic hepatocytes).
  • the resulting suspension was filtered through two layers of nylon fabric, followed by twice washing with Hanks saline.
  • the initial viability of the isolated hepatocytes was determined after receiving a suspension of fetal liver cells in a test with 0.1% trypan blue solution using light microscopy. Then, the number of fetal liver cells was counted in 1 ⁇ l in the Goryaev chamber and the resulting suspension of fetal cells was placed in an optimized nutrient medium in bottles containing 50 ml of medium at the rate of 1x10 6 hepatocytes per 1 ml. In parallel with the placement of fetal hepatocytes in the nutrient medium, samples were taken for bacteriological control (culture sterility test) and the blood type and Rh factor of the fetus were determined, which were taken into account when selecting the recipient. The resulting suspension was cultured in vitro from 1 to 5 days in various nutrient media: Fetal human serum (FSC); Hanks solution, Needle medium with the addition of L-glutamine; modified medium 199 (according to the original method).
  • FSC Fetal human serum
  • the proliferation index the ratio of the number of cells grown to the number of seeded cells.
  • dexamethasone 0.05 mg / ml
  • Hepatocyte viability at different stages of cultivation was determined by morphological examination. For 1 day on RPMI medium 1640 + 30% of single-group plasma + 10% of mediators of fetal hepatocytes, from the beginning of cultivation in the culture there are liver cells and hematopoietic elements at different stages of differentiation. Hepatocytes in culture are represented by clusters of polygonal cells with large nuclei with a significant amount of euchromatin containing up to five nucleoli. Among hepatocytes, cells containing two nuclei are noted, basophilic inclusions are present in the cytoplasm of cells.
  • hepatocytes form cell aggregates (up to 25-30 cells) resembling hepatic beams.
  • the culture contains spindle-shaped fibroblast-like cells containing small, elongated, intensely stained nuclei.
  • the number of hematopoietic cells continues to decline; by 6 days of cultivation, hepatocyte cell aggregates with light nuclei and basophilic inclusions in the cytoplasm are preserved in the culture.
  • 7 days of cultivation around accumulations of hepatocytes. When cultured in the absence of growth factors, hepatocytes remained viable in the culture for no more than 5-6 days.
  • the described method for culturing embryonic liver cells allows to obtain a sufficient number of hepatocytes in a short time, to reduce the number of hematopoietic ballast cells in cultures, and to significantly increase the hepatocyte survival time in culture.
  • Single cytochemical inert fetal hepatocytes at different stages of mitosis are determined in liver prints, most of which is diluted chromatin.
  • the described method for cultivating human fetal hepatocytes cells allows one to obtain a sufficient number of cells in a short time, to reduce the number of hematopoietic ballast cells in cultures, and to significantly increase the hepatocyte survival time in culture. During the cultivation period, it becomes possible to conduct a test for bacterial and viral contamination of cellular material in order to ensure the safety of transplantation.
  • hepatocytes were to be cultured, and after cryopreservation with a test for determining cell viability, by supravital staining with 0.1% trypan blue solution, the initial cell viability was determined (93.0 ⁇ 2.0% - whole, 4.7 ⁇ 2.7% - holonuclear, 2.3 ⁇ 1.7% - destroyed).
  • the next step was to remove the hepatocyte culture by standard methods and pour them into sterile cryovials with the addition of a sterile glycerol solution in a ratio of 10: 1 and then undergo 5 stages of low-temperature freezing
  • Hepatocytes to be lyophilized are grown under optimal conditions before the start of the stationary phase of growth or the end of the cultivation process in a nutrient medium; growth should be plentiful. Then the cells or, respectively, resting forms are suspended in special fluids. protective environments.
  • the composition of the protective environment includes various substances that protect cells from damage in the period of freezing and drying.
  • the density in the protective medium should be as high as possible -10 9 -10 10 cells in 1 ml.
  • the resulting suspension is poured into ampoules or into 1 ml penicillin vials of neutral glass, frozen at a temperature of -20 ° to -70 °, then dried on a Bench Top freeze dryer (Virtis, USA).
  • the residual moisture of lyophilized cells ranges from 1-6%. Storage at a temperature of +4 provides longer storage for up to 8 months (Figure 17.18).
  • Table 1 The ratio of the number of studied fetuses and gestational age.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Virology (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Immunology (AREA)
  • Nutrition Science (AREA)
  • Physiology (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne la médecine et notamment la thérapie intensive. Ce procédé est destiné à extraire qualitativement et quantitativement des hépatocytes du fœtus humain utilisés pour la transplantation à l'intérieur d'organes et utilisés dans le traitement combiné de malades souffrant de maladies du foie, y compris les maladies chroniques. L'invention consiste en ce que dans le procédé de production d'hépatocytes du fœtus on utilise le foie du fœtus obtenue après l'interruption de grossesse pour des raisons sociales après une durée de 18 à 22 semaines. Les hépatocytes du fœtus sont défragmentés, cultivés, cryoconservés et lyophilisés. L'application de ce procédé à la pratique médicale permet de l'utiliser largement pour traiter les patients souffrant de maladies hépatiques, y compris dans des formes chroniques, et d'attendre jusqu'à ce que leur tour en matière de transplantation arrive. Le procédé est assez efficace et peut traumatisant.
PCT/KZ2011/000009 2011-05-11 2011-05-11 Procédé de production de lyophilisats à partir d'hépatocytes humains Ceased WO2012154016A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/KZ2011/000009 WO2012154016A1 (fr) 2011-05-11 2011-05-11 Procédé de production de lyophilisats à partir d'hépatocytes humains

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/KZ2011/000009 WO2012154016A1 (fr) 2011-05-11 2011-05-11 Procédé de production de lyophilisats à partir d'hépatocytes humains

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WO2012154016A1 true WO2012154016A1 (fr) 2012-11-15

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2781448C1 (ru) * 2021-08-18 2022-10-12 Федеральное государственное бюджетное учреждение науки Иркутский научный центр Сибирского отделения Российской Академии наук (ИНЦ СО РАН) Фармацевтическая композиция для получения лиофилизата культуры клеток печени, обогащенного фактором роста гепатоцитов
EP4257142A1 (fr) 2022-04-08 2023-10-11 Saparbayev, Samat S. Procédé de traitement de lésions hépatiques chroniques diffuses par transplantation intraveineuse de cellules souches
DE102023121794A1 (de) 2023-08-15 2025-02-20 Yedil Bekbayevich Kurakbayev Dosierungsschema einer Stammzellsuspension zur Behandlung chronischer diffuser Lebererkrankungen

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2116793C1 (ru) * 1995-05-17 1998-08-10 Зозуля Андрей Александрович Способ получения комплекса веществ, обладающих противовоспалительным, ранозаживляющим действием, ингибирующим перекисное окисление липидов, оптимизирующих функциональное состояние тимуса у старых животных
RU2160112C1 (ru) * 2000-04-10 2000-12-10 Сухих Геннадий Тихонович Способ приготовления клеточного трансплантата из фетальных тканей
RU2272638C1 (ru) * 2004-08-18 2006-03-27 ЗАО "РеМеТэкс" Биотрансплантат, способ его получения и способ лечения хронического гепатита и цирроза печени (варианты)

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2116793C1 (ru) * 1995-05-17 1998-08-10 Зозуля Андрей Александрович Способ получения комплекса веществ, обладающих противовоспалительным, ранозаживляющим действием, ингибирующим перекисное окисление липидов, оптимизирующих функциональное состояние тимуса у старых животных
RU2160112C1 (ru) * 2000-04-10 2000-12-10 Сухих Геннадий Тихонович Способ приготовления клеточного трансплантата из фетальных тканей
RU2272638C1 (ru) * 2004-08-18 2006-03-27 ЗАО "РеМеТэкс" Биотрансплантат, способ его получения и способ лечения хронического гепатита и цирроза печени (варианты)

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
POKROVSKOGO, M.: "Entsiklopedichesky slovar meditsinskikh terminov, pod redaktsiei", MEDITSINA, vol. 1, pages 836, 909 *
SKOROBOGATOVA N. G. ET AL.: "Osteogennye i agipogennye svoistva fibroblastopodobnykh kletok-predshestvennikov fetalnoi pechenii cheloveka", TSITOLOGIA, vol. 50, no. 4, 2008, pages 317 - 322 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2781448C1 (ru) * 2021-08-18 2022-10-12 Федеральное государственное бюджетное учреждение науки Иркутский научный центр Сибирского отделения Российской Академии наук (ИНЦ СО РАН) Фармацевтическая композиция для получения лиофилизата культуры клеток печени, обогащенного фактором роста гепатоцитов
EP4257142A1 (fr) 2022-04-08 2023-10-11 Saparbayev, Samat S. Procédé de traitement de lésions hépatiques chroniques diffuses par transplantation intraveineuse de cellules souches
DE102023121794A1 (de) 2023-08-15 2025-02-20 Yedil Bekbayevich Kurakbayev Dosierungsschema einer Stammzellsuspension zur Behandlung chronischer diffuser Lebererkrankungen

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