EA009326B1 - Method for testing quality of cell culture - Google Patents

Abstract

The invention relates to area of cell technologies, namely to the analysis of cultivated in vitro cells, and can be used in biotechnology, cosmetology and medicine (cell transplantology) for production of the characterized cells possessing medical or cosmetic properties. The method for testing quality of cell culture includes mass spectrometry analysis of cell surface proteins. Washed living cultivated cells are treated with nonkilling for cells concentration of protease followed by collection of peptides, released from cell surface proteins, and their molecular weights determination by mass spectrometry analysis. To control presence of qualitative characteristics of investigated cell culture the set of the obtained molecular weights compares with the set of molecular weights obtained by the same way from other living cells with known qualitative characteristics. The technical result, reached by using of the patented invention, consists in increase of objectivity (accuracy) of cultivated in vitro cells identification with required useful properties at decrease of time and material inputs.

Description

The invention relates to the field of cellular technology, namely the analysis of cultured ίη νίίτο cells and can be used in biotechnology, cosmetology, medicine (cell transplantation), to obtain a characterized cellular material with therapeutic or cosmetological properties.

The problem of analyzing cellular material, namely, controlling variability, is an urgent task in all areas where cultured cells are used. So, experiments on cell cultures are an integral part of research in biology and medicine. On cellular models, such biological processes as apoptosis, differentiation, oncological transformation of cells are studied, many aspects of metabolism are studied, mechanisms of drug action are revealed, etc. Recent advances in cell technologies allow cultivated cells to be used as drugs that are promising, in the opinion of many scientists, in the treatment of many previously incurable diseases (Tetkkkky Α.ν. е1 а1., Matshayap k1et se11k, ReLay. Rec., 2006, ν. 59 , 13-20).

However, the phenotype of cultured cells is variable. The transfer of cells from physiological conditions ίη νίνο into culture flasks leads to cell stress and a significant reorganization of their metabolism. The reason for this is the impossibility of reconstructing in the test tube a habitat that is natural for cells ^ pdj νΗ., 8ау IV., H1k1opsa1 c1a1tk apb siggep11p (Ergre1a11op5th gerNsa (1me adtd, No. 11. Vytsesypo1., 2002, ð.),.....,........) .29............., 17. n. Segknop N., Segknop Ό., Cr111s1 akkektep! APT 1Ttoa11H1yup, ίη: §1e1n C.8., Vaquegda Α., Syugbapo Α., EepNagb! Ό.Τ. (ebk.). , No.e \ t Watk, 1999, pp. 348-373; Cr1k1oGa1o ν.1. Her a1., Lde-berebbeptobyu akopk o £ depe ehrtekküp ίη Nitap yblo1ak1k, SgN Ret. Eikatois Sepe Ehrtekkyup, 1998, ν. 8, 43-80);

activation of lysosomal hydrolases (WouteiLet K. et al a1., Nitapkt G1bogo1ak1k ίη νίίΐΌ b1Ggepn (1a a1opda att1n1e1epeade, Proc. No. 11a. Asab. δα.

degradation of hormone-dependent metabolism, shifts in the isoenzyme composition (Crjb ν.ν., Roerke Stürk and Steerecke Steerecke, Stroke Syndicr. , 303-307; Roerke 1.E., Crjb Ό.ν., Gockeh Kekhia1 bietepäkopoi ha (Neralosilek ίη kNogNogsi111e, L1sóNo1 ^ Μirr1., 1987, ν. 1, 263-264);

changes in the synthesis of glutathione (Retetk 1.1.1t. it a1., Electricity of the Synthetic Heart and Lyupe bycococcus ikeb yogi Le Rakkardsh oi Negenec syiiteb sy11k, Tohyo1. iue., 2000, η.

the loss in the process of cultivation of extracellular proteins and the degradation of the receptor apparatus (Niddshk IV. a1.)

the increase in cells with each passaging of telomerase activity and the shortening of telomeres (Wobapag ί. her a1., Exilecropsy 1st crap byybyscopic 1e1tegake 1n, 295, 349, 34, 349, 35. ultimately, leads to the inability of cells to proliferate (Nauyuk L., MooLeab R. 8., THe capa SIAAiop oi Nitap b1p1o1b c 11 kyatk, Exp. Ce11 Rec., 1961, ν. 25, 585-621);

loss of the histocompatibility complex (SSCPK T. e1 a1., Lokk oi ί сиη sy11ite, Proc. №111. Αsub. δα. υδΑ, 1986, ν. 83, 446-450; Αη. R., e1 a1., Expdtekkyp oh ta) About Mr. Sokolosov (MHC) Adedek apb Ley 1okk Syndiigi гепη gepa1 sagstota, Eig. 1. Sapseg, 1993, ν. 29, 2158-2160).

Thus, ίη ν yo cells are susceptible to morphofunctional degeneration, leading to their death. Moreover, the existing абилиη νί destabilizing conditions and the natural selection of cellular material lead to the emergence of a spectrum of different cell populations.

In medical cell technology, the issue of cell culture variability is most acute. Take, for example, the creation of cancer vaccines based on cultured tumor cells of a patient. Under cultivation, the antigenic phenotype of the cells ^ pd ^ P., e1 a1., Exprtkkup oi sha) og rykUsotraiPNu co-worker (MNS) aidepk apb Ley 1okp opiigpe ίη hepa1 sagstota, Eig is significantly distorted. 1. Sapseg, 1993, ν. 29, 2158-2160; No. APS. 1. E1 a1., Si11igeb sotrokye kkt dikayk: 1y1odya1kkt ec. | Iya1e11k repkShpd taksi ekhrapkyup, bansa1, 1989, ν. 22, 191-193), which leads to a mismatch of vaccine antigens and tumors. This is probably due to the fact that in most of the randomized trials of such vaccines conducted in the last decade, no obvious antitumor activity was obtained (ENHR.1. E1 A1. NoHapa Sapseg 1pcLie gapbotpheb sišsa 1p1.TypeOut.ToOf.Type.Type.Type.Type.Type.Type.Here, 1981, ν.61, 1267-1277; Mysye 11 M.8. ., 1998, ν. 25, 623-635).

No less relevant is the issue of cell variability when using cell transplants obtained by reproducing the cells of the patient himself. In particular, there are known methods for treating autologous fibroblasts (see, for example, Biotransplant and a method for correcting soft tissue defects, a method for producing a biotransplant, application: 2005127087/15, publ. 20.08.2006, IPC А61К35 / 36; А61К35 / 36, as well as ν09840027 , publ. 17.09.1998, IPC ПК61Α2 / 10; Α61Ε27 / 24; Α61Ε27 / 38; C12№5 / 00; υδ5660850, publ. 26.08.1997, IPC C12№5 / 06; Α61Ε2 / 00). Change in the process of cultivating anti

- 1 009326 genes (SoShp8 T. e! A1., Hock oG NLL ίη Siyige, Proc. No. 11. Asab. 8c1. I8A, 1986, ν. 83, 446-450) or, for example, the loss of the ability to proliferate (NauPuk ., Mogeyeab R.8., Tye kepa1 SyiVayop oG Yitap B1R1o1b se11 kishpk, Exp. Se11 Kek., 1961, ν. 25, 585-621) makes such biotransplant nonsensical, and, possibly, harmful.

Thus, cultured cells used in medical and cosmetic preparations must be subjected to qualitative analysis without fail.

Today, the assessment of the quality of cultured cells includes methods aimed at identifying contamination, determining the number of living cells, their tissue affiliation, identifying cross -contamination (contamination with cells of another type), as well as their absolute identification.

There are various methods for detecting contamination of cellular preparations with mycoplasma, fungi and viruses. These methods allow to characterize the quality of the cellular preparation, but do not characterize the quality of the preparation cells themselves.

There are various methods for assessing the quality of culture, based on the exclusion of the dye by living cells, the effectiveness of cloning and proliferation in mass culture (Hay R. Preservation and quality assessment of cells, in the book: Animal Cell Culture, ed. Z. Freshni, M .: Mir , 1989, pp. 108-164). These methods allow only to determine the percentage of living cells in the cell preparation and their viability, but do not give an idea of the qualitative (special) characteristics of the cells themselves.

There are various methods for determining tissue affiliation of cell lines, namely based on a thin structural analysis of cells using an electron microscope; immunological test of cytoskeleton proteins (Katahegk E.S..8. e! a1., Co1b 8rppd Nagyog 8utr. Oiap1. Vyu1, 1982, ν. 46, 331); detection of tissue-specific antigens (No.) \ 1 p.1. apb Mi1beg KER., 'Tbejpaysayop og ok! eosu! ek ίη ok! eo1a! -11ke se11 sy1! igek iktd a topos1opa1 ubybobu krest1psau byyes! eb adatk! ok! eos! ek, 1986, ν. 84, 342-347); biochemical testing of specific cell functions (applicable to cells with specific functions, see, for example, Naw K.1. e! a1., Atepsap Toure C1! above So11es! Yup Ca! iodie II, 4 ' ¹ ' ebp., 1983, Voskuse, MO, p. 12), quantification of immunological products (applicable for identification by hybridomas).

The lack of tests for tissue specificity in the evaluation of cellular preparations is the inability to determine the source of cells. In particular, fibroblasts used in cellular preparations can be of both fetal (embryonic) origin and isolated from adult tissues, for example, from skin or fatty tissue (adipogenic fibroblasts). Thus, fibroblasts obtained from various sources, although they belong to the same type of tissue, have different beneficial qualities.

Currently, there are several ways to identify cross-contamination and identification of cell cultures.

A known method of indirect cell staining with fluorescently-labeled antibodies. This method allows you to verify cell culture, to determine cross-contamination, to establish the variability of certain antigens (8! 1) C.8. W: Sop! Attat! Jup t T1kkige Si1!, Yedy 1. (eb.), Asabetiu Rgekk, 1973, Bopbop App #? Vogk, p. 1). The disadvantage of this method is the need to pre-receive species-specific antibodies for a particular culture, which are obtained by immunization of the animal with the test cells. The method can not characterize the loss of useful properties of cells in the process of cultivation.

A known method based on the analysis of isoenzyme composition. It has been shown that the determination of seven enzyme isoforms by electrophoresis reliably makes it possible to determine the identity of cells to a human (O'Veppe 8.1. E! A1., Ephute rototropyclic acypes c1dpa ektyt yitap se11 siyigek, 8c1epsa, 1977, ν. 195, 1345-1348 ). The disadvantage of this method is the duration of its execution (electrophoresis is 16-18 hours) and the applicability is only for detecting interspecific cross-Kontaminatsii. The method can not characterize the loss of useful properties of cells in the process of cultivation.

Known cytogenetic (karyological) method for assessing the presence of impurities of other cell types in culture. The method is based on the fact that chromosome sets can differ significantly in cells of different species, which can be detected using a microscope. However, if there is contamination by cells of a closely related species, the analysis becomes much more complicated (C-segmentation is used, see, for example, “B”! M., A Gar1b bcbcd 1ccc. | Ie Gogh yythyphococotec, Cusse !, 1971, ν.2, 971-972) . The method allows to detect only interspecific cross-contamination and can not establish the loss of useful properties of cells in the process of cultivation.

The immunological test for blood group antigens is known (Nau K.1. 1p: Magkek SoGoSeSe11, Heygapayop, Wytapk 8.K., Makyotaypo A.1. (Ebk.), 1984, Katep Rgekk, No. \ ν Wogk, p. 3) and histocompatibility antigens (Ro11ask M.8. E! A1., HbAA, B, C apb ΌΚ ayoapidep exhrgekküp op Goyu-kgkh siyyshbyt yitap! Total sec 11ypek, 1. No.11. Sapseg 1pk!., 1981, ν. 66, 1003-1012). The disadvantage of this method is the partial or complete absence of expression of these antigens in some cell cultures.

The use of polymerase chain reaction and DNA sequencing is known (LU MU e a1, 'Tbeycayyop apbaiyypysayop og atta1se11 siyige byy ro1utegake syat geacyopatryysyop apb ΩΝΛ

- 2 009326

5ss. | Ispstd. Ιη Uigo Ce11i1ag & Osus1orsp1a1 Vu1odu - Lesta1, 2003, ν. 39, 424-427; Ragob1 V. e1 a1., Zreaek ίάοηΐίΠοαΙίοη api paratigyop ok yitap apb attat1 se11 ípek: a RSK-акakeb yo6, Vu! Esysdiek, 2002, ν. 32, 432-434, 436, 438-440). The disadvantage of the method is the applicability to cell lines of non-closely related species, as well as the duration of execution, because he requires electrophoresis. The method can not characterize the loss of useful properties of cells in the process of cultivation.

DNA fingerprinting is known (see, for example, I82005123947, publ. 09.06.2005; ^ 02005116257, publ. 08.12.2005; I82006035261, publ. 16.02.2006). DNA is fragmented by a restriction enzyme, and the resulting DNA fragments differ in size and collectively form a unique profile to identify the source of the DNA. A similar approach is widely used to identify cellular material. DNA fingerprinting for the most commonly used restriction fragment length polymorphism (Kebra) (see., E.g., E. Kap1et e1 a1., Ca. Apa1ukk ge81g1s (1op GgadteSh 1epd! D u roKtogrYktk beohupopisShs as1b (ΌΝΑ) gesomegeb kgot bpeb 1oobk! CCA 1 . Eogepkyu 8s1 1986, v. 31, 403-408), the variable nucleotide tandem repeats (ΥΝΤΚ8) (ViboMe V. e1 a1., Apa1ukk ok! ye ΥΝΤΡ 1osik Ό1880 Ly 1ye RSK. Go11o \ us6 Ly yschy gekoyPyup-race, At. 1. Nite. Sep.!, 1991, ν. 48, 137-144) or microsatellites (1kkgeuk AD. E! A1., NuretauPa'T1shka! E1y! E 'teDopk sh yitap ΌΝΑ, No.! Ige, 1985, ν. 314, 67-73). With the exception of mono zigo This method allows to accurately identify the identity of the cellular material to the individual, however, the method does not characterize the change in the quality of the cell culture during the cultivation process.

A two-dimensional protein electrophoresis is known, which allows to detect up to 2 thousand proteins (Apbegkop Ο.Ο., Apbegkop .Ε., T \ tschuchoukk ca 1 \ uo-bnepküp11troiogyog11k: cancer, protrusion, apycchimus, Elysteraerosus jérégéryne-jérégérynekégé jérygérykéké jérégérygékéké et al. 1996, ν. 17, 443-453). The disadvantage of this method is the complexity of execution. The method requires the successive conduct of two electrophoretic separation of proteins, and therefore is not used as a routine method for assessing the quality of cell cultures. Moreover, two-dimensional protein electrophoresis does not characterize the fraction of cell surface antigens that is most relevant for cell therapy, since membrane proteins are not analyzed by this method.

It is known the use of mass spectrometry for the identification of cell cultures (2yapd X. e! A1., 1bl11Dsa! Juc ok Matshayap Se11 Biniktkd MABO1-T0P apb bc-E81-M8 / M8 Mack 8resploti, 1. At. 8os. Mack 8res! Go ., 2006, ν. 17, 490-499). A quick and relatively simple method for profiling cell proteins is described for mammalian cells by directly analyzing time-of-flight (MABN1 Τ0Ε) and other types of mass spectrometry. Using MABN1 mass spectra as a unique fingerprint, mammalian cell lines can be differentiated.

The disadvantage of this approach is low specificity, caused by the presence in the analyzed sample of many cytosolic (intracellular) proteins, whose fragments, as a rule, are not specific for cell types, clog up the mass spectra and do not allow sensitive analysis and characterize cells according to the most relevant for cellular transplantation membrane proteins.

From patent \ UO2006115426 (Method for regulating biochemical processes and device for its implementation, publ. 02.11.2006, IPC С01К23 / 16; С12М3 / 00; 02 ;1 / 00), there is a known method for assessing the quality of cultivation of cell cultures based on the registration of time intervals of cell growth reproduction and accumulation-mutation, in which the boundaries of the intervals are determined by radio signals in the mid-frequency and low-frequency ranges. The disadvantage of this method is the need to use special equipment, as well as the ability to determine parameters only as a result of constant monitoring of cell growth, which is not applicable to assessing the quality of finished cellular preparations.

A known method for evaluating the properties of a cell culture of neurons (see patent I82004106101, publ. 03.06.2004, IPC Ο01Ν33 / 487; С06Д10 / 00; Ο01Ν33 / 487) based on measuring the electrical activity of cells by growing them on a microelectrode chip. This method applies only to the culture of neural cells and does not apply to other cell cultures. A method of certifying chondrocyte cell culture is also known (see Patent 002095399, publ. 28.11.2002, IPC С12Ц1 / 42; 001Ν33 / 50; Ο01Ν33 / 68). The disadvantage of this method is its applicability only in regenerative therapy of cartilage tissues.

There is a method of determining the quality of cell culture, including mass spectrometric analysis of organic cell molecules. The known method involves the use of mass spectrometers that measure the mass of molecules with high accuracy through the use of ion-cyclotron resonance, which allows to increase the amount of information obtained from cell samples (I86974702, publ. 02.06.2005, IPC 601-227 / 62; 601Ν33 / 15; 601Ν33 / 483).

The disadvantage of this method is, on the one hand, the contamination of the spectra obtained by uninformative cytosolic proteins and other biopolymers, since whole cells are used for analysis, and, secondly, the use of a specific and expensive mass spectrometer, which allows determining the masses of molecules with high accuracy. In this regard, this approach is not widely used as a routine method for analyzing the quality of crops.

The closest analogue of the claimed invention is a method based on mass spectrum.

- 3 009326 analysis of exactly the surface proteins of cells, disclosed in the patent of RteratDop about £ дDYu-ripyeb p1acta tetbapeck (№003025565, publ. 27.03.2003, MPK-7 Ο01Ν27 / 62; С07К1 / 22; С07К16 / 28), in which method for determining the quality of cell culture, including mass spectrometry analysis of pre-isolated membrane proteins.

The disadvantage of this method is the necessity of using antibodies specific for membrane proteins or their antigen-binding fragments for the formation of complexes with membrane proteins for their subsequent isolation and purification. Such a multistep prevents the use of this approach as a routine method for analyzing the quality of cultured cells.

In the present invention, the task was to develop a new method for determining the quality of cell culture, focused on the analysis of cellular preparations, the distinguishing features of which are the speed of execution (not more than 2 hours), which will allow it to be used to assess the quality of finished cellular preparations before their application, given the short period their shelf life from the date of manufacture (several hours);

sensitivity, allowing to determine the loss of quality by the cells during cultivation;

sensitivity, which allows differentiation of morphofunctionally identical cells from one donor, but having different useful properties when applied;

focus on the analysis of cell surface proteins, the determination of the safety of which in the process of cultivation is most relevant;

integrability into the cultivation of cells, which implies the absence of special equipment and complex manipulations for the preparation of samples for analysis;

relative cheapness, so that the cost of analysis is incomparably lower than the cost of the cellular preparation;

minimal or no cell consumption, which will allow repeated analysis of cultured cells without their consumption;

the possibility of numerical expression of the results of the analysis, which is necessary for the implementation of the standard MMP, which excludes the human factor when assessing the quality of cell culture.

The technical result achieved when using the patented invention is to increase the objectivity (accuracy) of identifying cultured ίη νίΐτο cells for the selection of quality (with the expected useful properties) cells while reducing time and material costs.

This technical result is provided when implementing the method for determining the quality of cell culture, including mass spectrometric analysis of cell surface proteins. According to the present invention, the pre-washed living cells of the cell culture under study are subjected to the vital effect of protease, splintered fragments of surface proteins are selected and their masses are spectrometrically measured, the presence of qualitative characteristics of the cell culture under investigation is monitored by comparing the aggregate masses obtained with the mass spectrometrically determined masses of the surface fragments proteins of living cells with known qualitative characteristics.

In a preferred embodiment of the invention, trypsin is used as a protease.

To identify the relationship of cell culture to a specific group of cellular preparations with useful properties, control the availability of qualitative characteristics that determine the source of cells.

In a preferred embodiment of the invention, the availability of qualitative characteristics that determine the suitability for antitumor immunotherapy is monitored.

To establish the preservation of the useful properties of the cells, they control the presence of qualitative characteristics indicating that there is no change in the phenotype of the cells of the cell culture being studied during the cultivation process.

Primary cell culture can be used as living cells with known qualitative characteristics.

As living cells with known qualitative characteristics, cells that have not undergone changes in the phenotype during storage and culture can be used.

The cleaved fragments of surface proteins can be deglycosylated before mass spectrometric analysis.

Treatment of the primary cell culture with the vital effect of trypsin results in splitting off from the cell surface protein fragments, the composition of which is specific for specific cell cultures. Thus, according to this invention, the action of trypsin, as well as other proteases, leads to the release into solution of cultured cell-specific fragments of surface proteins, the totality of the measured masses of which is characteristic of the cell culture. For such characterizing cultured cells of mass sets, the term proteomic footprint (protein imprint) was introduced.

The cells are not killed by treatment with the vital effect of protease, which makes it possible to avoid contamination of the analyzed samples with major proteins of the cytoplasm of the cell and to avoid the consumption of glue

- 4 009326 exact material in the analysis.

Further, the invention is illustrated with specific examples of implementation of the invention and the attached figures, which depict the following:

in fig. 1 is a preparation scheme for a proteomic footprint of cultured cells according to the invention;

in fig. 2 - proteomic footprints of fibroblasts from various human tissues, classified by hierarchical cluster analysis;

in fig. 3 is the mass spectrum of fragments of surface proteins and the corresponding footprint obtained according to the second embodiment of the invention from a newly initiated primary culture of human tumor cells (3A); the mass spectrum of the surface protein fragments and the corresponding footprint obtained from the same cell culture after a brief culture thereof (3B); the mass spectrum and the corresponding footprint obtained from the same cell culture after a longer cell culture (3B).

Example 1. On human fibroblasts.

Morphologically, all types of fibroblasts are almost identical (have a spindle shape). However, fibroblasts obtained from various sources differ significantly in their properties and, accordingly, have different indications for use in cell therapy.

In particular, fibroblasts derived from human adipose tissue are distinguished by pluripatency like stem cells (see, for example, RA 2c1 a1., Nitap abroc Nkkie 1k and coigse oh ιηιιΐΐίΐΐίροίοηΐ c1et ce11k, Mo1. Βΐοΐ. Ce11. 2002 , ν. 13, 4279-4295). Fibroblasts of human skin have a weak potential for reproduction, but are suitable for mesotherapy with large doses of reproduced cells of the patient himself. Human embryonic fibroblasts have good reproduction potential and are suitable for allogeneic cell transplantation (Sukhikh GT, Fetal Cell Transplantation in Medicine: Present and Future, Bull. Exp. Biol. Med., 1998, 126, Appendix 1, 3 -13). Fibroblasts of the dermal papilla of the hair follicle have trichogenic properties and can be used in cell therapy of alopecia (see, for example, Biotransplant patent, method of its production (options) and method of treatment of alopecia, application: 2004125092/15, publ. 08.18.2004).

Below is an example of determining, according to the invention, the qualitative characteristics of a fibroblast culture under test by establishing the identity of its proteomic footprint to similarly obtained footprints of fibroblast cultures of known origin and with established useful properties.

Forty-two primary cultures of dermal, adipogenic, fetal fibroblasts and dermal papilla of the hair follicle fibroblasts were obtained from appropriate sources according to previously described methods. Primary cultures of cutaneous fibroblasts were obtained according to the method of KtShe and Ikyet (1 st 11a apb syiite οί kksh G1bot1ak1k, Mebyubk Mo1. Meb., 2005, ν. 117, 83-98). Primary cultures of adipogenic fibroblasts were prepared according to Method 2ic et al. (MiNspeade sekk Ggot Nitap abrokke ykkie: pprysa Tsopk Gog se11-lakeb 1styrlek, T1kkie Epd., 2001, ν. 7, 211-288). Primary cultures of fibroblasts of the dermal papilla of the hair follicle were obtained according to the method 2HPD-Ha et al. (Vyuyoduya1 syagasYupkhNop og sp11pegebegta1 rarSa se11k apb Nag1 Gos1e tdepegigiop shmNgo apb uAo, Supermarket Meb1sa1oitpa1, 2006, ν. 119, 275-281). Primary cultures of human fetal fibroblasts were obtained from abortive material according to the method of 8aCalop et al. (ReEushb uesti-teb1a1e depoe 1gapkGag1n1o Nitap rtagu tuodeshs se11k 1eabk 1o ehrggykup tiks1e Pegkshu uy, Nit. Opepe TiN., 1993, ν. 4, 713-723). The cultivation of primary cultures was carried out under identical conditions (growth medium DMEM, 5% CO ₂ , 37 ° C, 10% fetal bovine serum). On days 20-25 after the initiation of cultures, a sterile cell preparation (1 million fibroblasts in 1 ml of physiological saline) was made, which was analyzed according to the following protocol.

1. The cell preparation containing the tested fibroblasts is poured into a culture flask under sterile conditions. The growth medium (DMEM with 10% fetal bovine serum) is added to the vial and incubated at 37 ° C in a CO ₂ incubator for 30-45 minutes.

2. Remove the growth medium from the vial and wash the cells attached to the bottom of the vial three times with sterile saline using a volume equal to half the growth medium.

3. Add a 0.0001% trypsin solution to the cells using 1 ml of solution per 25 cm of the surface of the culture flask.

4. Incubate the bottle at 37 ° C. Between the 5th and 7th minutes of incubation, trypsin solution containing protein fragments split off from the cells is removed from the vial.

5. Trypsin solution, selected from cells, is used for mass spectrometric determination of the aggregate mass of surface protein fragments corresponding to the analyzed cells.

6. The measured mass of fragments of the surface proteins of the analyzed cells is compared with the mass of the fragments of cell culture surface proteins with similar qualitative characteristics obtained by a similar method.

Mass spectrometry analysis is carried out as follows. Samples obtained in the process

- 5 009326 incubation of cells with trypsin (see p. 4 of the protocol), are desalted using tips for automatic pipettes with reversed phase ΖίρΤίρ ₍ · ₈ (M1 Shrot Sogr., USA), in accordance with the manufacturer's protocol. The masses of peptide fragments contained in assay, measured by time-of-flight (MAYSH-TOR) mass spectrometry. 2 µl of desalted solution is mixed on a 1: 1 mass spectrometry target with a saturated solution of a-cyano-4-hydroxysinamic acid containing 50% acetonitrile and 0.5% trifluoroacetic acid. on the target, droplets of samples are dried in air and mass spectrometry analysis is performed in the range of peptide masses from 600 to 4000 Da.

The obtained mass spectra corresponding to different cell lines are pretreated for subsequent classification by hierarchical cluster analysis. The list of peptide masses is transferred to the binary code (1- there is a certain peptide mass in the spectrum and, accordingly, 0 is the absence of peptide mass), thus generating the proteomic footprint of the cell culture. Classification (grouping) footprints is clusterized by the Ward method using squares of Euclidean distances to calculate the distance matrix.

From FIG. 2 shows that the footprints of 42 fibroblast preparations from various sources, such as the dermal papilla of the hair follicle, adipose tissue, adult forearm skin and fetal skin, are clearly classified into four groups according to the origin of fibroblasts (1, 2, 3 and 4 groups in FIG. 2, respectively). Thus, having a tested cell preparation of fibroblasts, one can accurately determine its origin by the belonging of its footprint to one or another cluster. For example, if a cell preparation under test corresponds to the one marked with an asterisk (*) in FIG. 2 footprints, it can be reliably argued that the fibroblasts of the test drug were isolated from the dermal papilla of the hair follicle, propagated accordingly, and in the process of cultivation, no qualitative characteristics were lost that made it possible to attribute these fibroblasts to fibroblasts with trichogenic properties.

If the test cell culture does not belong to any of the clusters (see, for example, the footprint of adipogenic fibroblasts in cluster 2 'in Fig. 2), it can be argued that the cells do not belong to any of the sources to be determined or during cultivation their phenotype has changed and it is no longer possible to associate them with high-quality cellular preparations of established origin and with known properties.

From the above example, it is clear that the set (set) of molecular masses of the surface protein fragments, the so-called. proteomic footprint, is a kind of bar code that characterizes cells for their relationship to fibroblasts with certain properties of interest to cell therapy.

It should be noted that sampling a trypsin solution containing fragments of surface proteins split off from the cells should be carried out until the cells are detached from the bottom of the culture flask under the influence of trypsin, which will prevent cells from entering the sample and avoid the additional stage of sample cleaning.

The conditions of protease treatment of cells are determined experimentally for each type of cell and the activity of the protease used, and they can vary significantly from 0.00001 to 0.05% for the protease concentration and from 30 s to 10 min for the treatment time of cells. In the case of using trypsin with a different activity, its concentration varies in direct proportion to the increase or decrease in activity.

In other embodiments of the invention, any other mass spectrometry is used.

Trypsin solution can be prepared both with sterile saline solution and with any suitable saline or buffer solution.

Instead of trypsin, other proteases can be used, for example, chymotrypsin, proteinase K, etc.

The cleaved fragments of surface proteins may be deglycosylated prior to mass spectrometry analysis, for example, pronase.

Instead of trypsin, a combination of proteases can be used.

In the case of suspension cell culture, it is necessary to precipitate cells to the bottom before sampling, for example, by centrifuging or use any other suitable method to remove cells from the test sample.

Desalting and concentration of the peptides of the sample for mass spectrometric analysis can be carried out in any suitable way.

In other embodiments of the invention, if the applied protocol for mass spectrometric analysis does not require desalting and / or concentration of the analyzed sample containing fragments of surface proteins, desalting and / or concentration is not carried out.

In the case of using trypsin to remove attached culture cells from the substrate, upon receipt of the cell preparation, prior to analysis, additional incubation of the cells under appropriate conditions is carried out for 18–20 h to repair the surface proteins by the cells.

In other embodiments of the invention are used to form the characteristic

- 6 009326 of the mass aggregate is not the appearance of one or another mass of the surface protein fragment, but a change in its intensity in the mass spectrum, which corresponds to a change in its concentration in the analyzed sample.

In other embodiments of the invention, any suitable mathematical processing of the measured masses of protein fragments is used, allowing to correctly classify cellular material according to the properties of interest, for example, using regression analysis, k-theapl clustering, neural network, XRD, 8UM, 8OM, etc., and also their combinations.

The patented method can be applied to any type of cell preparation, in particular, to cell suspension, mixed cultures, organotypic cultures and cell aggregates (granules, spheroids), as well as to freshly isolated cells and tissue fragments.

Example 2. On human tumor cells.

The invention is further illustrated by a second example of assessing the quality of a culture of human colon cancer cells for their suitability for antitumor vaccination.

The cells isolated from the patient’s tumor, cultivated and subsequently used for antitumor vaccination should be identical to the patient’s tumor cells. However, cell cultivation significantly distorts the phenotype of the primary culture due to the impossibility of artificially recreating the conditions under which the tumor cells grew in the patient's body. Thus, the quality of cultured tumor cells used for vaccination is subject to mandatory control and is expressed in the degree of their compliance with the patient's tumor cells. A protocol for establishing such compliance is presented below.

1. The growth medium is removed from the flask with cultured tumor cells and the cells are washed three times with 0.9% sodium chloride solution or phosphate buffer of silane, each time using a volume equal to at least half of the growth medium poured into the culture flask. As a result of cell washing, traces of serum contained in the growth medium must be removed.

2. To the cells add 0.0001% trypsin solution (activity ~ 3000 U / mg), using 1 ml of solution per 25 cm ^{2 of the} surface of the culture flask.

3. Incubate the bottle at 37 ° C. Between the 5th and 7th minutes of incubation, a solution containing fragments of surface proteins split off from the cells is removed from the vial. Tumor cells at the time of sampling should be attached to the bottom of the culture flask. If under the influence of trypsin, some of the cells detached and float freely, then the growth medium should be centrifuged at 400 d for 5 min and the supernatant, free of cell contamination, should be used.

4. To inactivate trypsin residues in the culture flask, add freshly prepared culture medium containing fetal bovine serum, and continue cell culture at 37 ° C and 5% CO ₂ .

5. The solution obtained in step 3 is analyzed by mass spectrometry and compared with the spectrum obtained in a similar way (claims 1–4), but earlier for freshly isolated tumor cells.

Mass spectrometric analysis is performed according to the protocol of mass spectrometry of glycosylated peptides (Ta.) W. M. e! a1., ”01ГГегеп11а1 апа1ук18 оГ кке-креПс д1усапк Отр ус О 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1η 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1η η 1η 1η т1 1ттт:: ар ар ар ар ар ар ар ар ар ар ар ар ар ар ар ар ар ар ар ар ар арГГй теб теб Gog d1usorerbbe eppsitepurne! 15, 13321340). This is explained, on the one hand, by the fact that extracellular protein fragments are, as a rule, glycosylated, on the other hand, it is glycosylated fragments of the surface proteins of tumor cells that are most immunogenic and are probable vaccine antigens (see, for example, Etapso A., CT-Wakeb Sapseg P ^ eνeηΐ ^ νe / Tye ^ areiΐ ^ with Uasshek Gog Satshotak: Colee Titoig-A88os1a! Eb SaghoNubShe Aidedek, 8sapb. I. 1ttipopo1., 2005, ν. 61, 391-397.)

140 μl of the analyzed solution of the surface protein fragments are mixed with 140 μl of ethanol, 720 μl of butanol and 15 μl of Sepharose CH4B are added. Incubate with slow stirring for 45 minutes. After incubation, the sepharose is washed twice with a solution with the same alcohol and butanol content and incubated for 30 minutes in a 50% ethanol solution. The ethanol solution is taken from Sepharose and dried on a rotary evaporator. The resulting precipitate is dissolved in 10 μl of water and analyzed by time-of-flight (MAB1-TOE) mass spectrometry. 2 μl of the analyzed solution is mixed on a mass spectrometric target in a 1: 1 ratio with a saturated solution of 2,5-dihydroxybenzoic acid containing 50% acetonitrile and 0.5% trifluoroacetic acid. The droplets of the samples obtained on the target are dried in air and mass spectrometry analysis is carried out in the peptide mass range from 1000 to 4000 Da.

According to the obtained data, at least 95 wt.% Protein fragments are reproduced in the repeated (control) mass spectrometric analysis of cell culture, which can be considered a criterion for the identity of two compared cellular preparations. It is proposed to evaluate the suitability of the cell preparation for vaccination of the patient based on the identity of at least 90% of the total mass of protein fragments in the analyzed cells and freshly isolated donor tumor cells.

FIG. 3A shows the time-of-flight spectrum of protein fragments and the corresponding footprint obtained according to the second embodiment of the invention from freshly isolated tumor adhesives.

- 7 009326 current of human colon cancer. FIG. 3B shows the mass spectrum and the corresponding footprint of the same cells propagated by cultivation (1st passage) and suitable for antitumor vaccination. The spectrum taken from them is identical to the original spectrum obtained from freshly isolated tumor cells. FIG. 3B shows the mass spectrum of the same cells propagated by cultivation (2nd passage), but no longer suitable for anti-tumor vaccination. In the process of cultivation, significant changes occurred in the surface proteins of the cells (see, for example, the 1800-2000 Da footprint in Fig. 3B), which allowed them to be considered nonidentical cells of the original donor tumor.

In case of using protease to dissociate tumor tissue in order to release tumor cells, it is necessary to carry out mass spectrometry analysis to obtain a sample of the footprint of the original (unchanged) cell culture no earlier than one day after the initiation of the tumor culture so that the cells can restore the surface proteins damaged during the isolation.

The set of masses of fragments of surface proteins is a kind of bar code, i.e. an indicator that most objectively characterizes cells.

Therefore, the control of the presence of qualitative characteristics of the studied cell culture by comparing the aggregate of obtained masses of fragments of surface proteins of its cells with a set of mass spectrometrically determined masses of fragments of surface proteins of living cells with known qualitative characteristics makes it possible to most accurately select qualitative (with the expected useful properties) cells.

The use of mass spectrometry in the implementation of the method allows one to determine the quality of the cell culture rather quickly - within not more than 2 hours. This will allow it to be used to assess the quality of the finished cellular preparations before their use, taking into account their short shelf life from the time of manufacture (several hours).

In addition, the obtained mass spectra can be transformed with the possibility of numerical expression of the results of the analysis, which is necessary to implement the standard SMR, which excludes the human factor when assessing the quality of cell culture, and will ensure the necessary objectivity of the results.

The accuracy of determining the presence of qualitative characteristics in the studied cell culture in the implementation of the patented method is also ensured by the use of living cells of the studied cell culture, which is subjected to the vital effect of protease. The cells are not killed by treatment with the vital effect of protease, which makes it possible to avoid contamination of the analyzed samples with major proteins of the cytoplasm of the cell and significantly reduce the consumption of cellular material in the analysis of cell culture.

Contamination of the analyzed samples also avoids pre-washing the cells, which is necessary to remove traces of serum contained in the growth medium.

Thus, the patented method allows us to characterize cells according to the most relevant, including cell transplantation, surface proteins.

Claims (8)

1. A method for determining the quality of cell culture, including mass spectrometric analysis of surface cell proteins, characterized in that the pre-washed living cells of the studied cell culture are exposed to vital proteases, cleaved fragments of surface proteins are selected and their masses are determined spectrometrically, and quality characteristics are monitored in the studied cell culture by comparing the totality of the obtained masses with the totality of mass spectrometrically determined mass fragments of surface proteins of living cells with known qualitative characteristics. 2. The method according to claim 1, characterized in that trypsin is used as a protease. 3. The method according to claim 1, characterized in that they control the presence of qualitative characteristics that determine the source of the cells. 4. The method according to claim 1, characterized in that they control the presence of qualitative characteristics that determine the suitability for antitumor immunotherapy. 5. The method according to claim 1, characterized in that they control the presence of qualitative characteristics indicating the absence of changes in the phenotype of the cells of the studied cell culture during cultivation. 6. The method according to claim 1, characterized in that the primary cell culture is used as living cells with known qualitative characteristics. 7. The method according to claim 1, characterized in that as living cells with known qualitative characteristics use cells that have not undergone changes in the phenotype. 8. The method according to claim 1, characterized in that the cleaved fragments of surface proteins are deglycosylated before mass spectrometric analysis.