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WO2008123793A1 - Procédé pour tester la qualité d'une culture de cellules - Google Patents

Procédé pour tester la qualité d'une culture de cellules Download PDF

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Publication number
WO2008123793A1
WO2008123793A1 PCT/RU2007/000571 RU2007000571W WO2008123793A1 WO 2008123793 A1 WO2008123793 A1 WO 2008123793A1 RU 2007000571 W RU2007000571 W RU 2007000571W WO 2008123793 A1 WO2008123793 A1 WO 2008123793A1
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cells
cell
cell culture
mass
fragments
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Russian (ru)
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Petr Genrievich Lokhov
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry

Definitions

  • the invention relates to the field of cellular technologies, namely to the analysis of cultured ip vitro cells and can be used in biotechnology, cosmetology, medicine (cell transplantology) to obtain the characterized cellular material with therapeutic or cosmetic properties.
  • biotechnology namely to the analysis of cultured ip vitro cells and can be used in biotechnology, cosmetology, medicine (cell transplantology) to obtain the characterized cellular material with therapeutic or cosmetic properties.
  • cosmetology cell transplantology
  • ip Steep GS, Vaserga A., Giordapo A., Dephardt D.T. (eds.). “The Molesular Basis of CeIl Susl apd Grövth Soptrol”, Wileu-Liss, New Yörk, 1999, pp.
  • in vitro cells are susceptible to morphofunctional degeneration leading to their death.
  • the existing in vitro destabilizing conditions and the natural selection of cellular material leads to the appearance of a spectrum of different cell populations.
  • the issue of cell culture variability is most acute. Take, for example, the creation of antitumor vaccines based on cultured tumor cells of a patient. During cultivation, the antigenic phenotype of cells is significantly distorted (Apgus R., et al., "Expresiop of major histocompotibilit complex (MHC) aptigeps apd th Sprintir loss on cultural ip repalcipoma", ⁇ ur. J. Sapser, 1993, v.
  • MHC major histocompotibilit complex
  • cultured cells used in medical and cosmetic preparations must undergo a qualitative analysis.
  • tissue affiliation of cell lines There are various methods for determining the tissue affiliation of cell lines, namely based on: fine structural analysis of cells using an electron microscope; an immunological test of cytoskeletal proteins (Ramakers F.C.S. et al., CoId Srripg Narbo Sr. Quapt. Biol., 1982, v. 46, 331); detection of tissue-specific antigens (Nij Weide PJ. and Mulder R. J.P.,
  • tissue specificity tests in evaluating cell preparations is the inability to determine the source of the cells.
  • the fibroblasts used in cell preparations can be either fetal (embryonic) in origin or isolated from adult tissues, for example, from the skin or adipose tissue (adipogenic fibroblasts).
  • adipogenic fibroblasts adipogenic fibroblasts
  • CS ip "Soptamiptiop ip Tissure Culture", Fog J. (ed.), Academis Press, 1973, Lopd add New York, p.l).
  • the disadvantage of this method is the need for preliminary obtaining species-specific antibodies for a particular culture, which are obtained by immunization of the animal with test cells. The method cannot characterize the loss of useful properties by cells during cultivation.
  • cytogenetic (karyological) method for assessing the presence of impurities of other types of cells in culture.
  • the method is based on the fact that chromosome sets can significantly differ in cells of different types, which can be detected using a microscope.
  • G-segmentation is used, see, for example, S Smithbright M., “ ⁇ parid b Economicspdi ⁇ g t survey ⁇ réelle ⁇ maneuver h ⁇ réelle ⁇ till”, Lapset, 1971, v.2, 971-972).
  • the method allows revealing only interspecific cross-contamination and cannot establish the loss of useful properties by cells during cultivation.
  • Known immunological test for antigens of the blood group (Nau RJ. In:
  • DNA fingerprinting is known (see, for example, US2005123947, publ. 06/03/2005; WO2005116257, publ. 12/08/2005; US2006035261, publ. 02.16.2006).
  • DNA is fragmented by restrictase and the resulting DNA fragments are different in size and together form a unique profile that allows you to identify the source of DNA origin.
  • a similar approach is widely used to identify cellular material.
  • RFLP restriction fragment length polymorphism
  • RFLP restriction fragment length polymorphism
  • VNTRS variable nucleotide tandem repeats
  • the known method involves the use of mass spectrometers that measure the mass of molecules with high accuracy due to the use of ion-cyclotron resonance, which allows to increase the amount of information obtained from cell samples (US6974702, published 02.06.2005, MTTK G01N27 / 62; G01N ⁇ / 15; G01N ⁇ / 483).
  • the closest analogue of the claimed invention is a method based on mass spectrometric analysis of precisely surface cell proteins, disclosed in the patent "R réelleraratiop schreibf highl- motivationurifikosd jackeriel réellem matterss m Economicsmbr Maisp réelles” (WO03025565, publ. 03/27/2003, MPK-7 G01N27 / 62; C07K1 / 27/62; C07K1 / 22/62; ; C07K16 / 28), which describes a method for determining the quality of a cell culture, including mass spectrometric analysis of pre-isolated membrane proteins.
  • the present invention was tasked with developing a new method for determining the quality of cell culture, focused on the analysis of cell preparations, the hallmarks of which are:
  • - sensitivity allowing to determine the loss of quality by cells during cultivation
  • - sensitivity allowing to differentiate morphofunctionally identical cells from one donor, but possessing various useful properties when applied;
  • the technical result achieved by using the patented invention is to increase the objectivity (accuracy) of identification of cultured ip vitro cells for the selection of high-quality (with expected useful properties) cells while reducing time and material costs.
  • the specified technical result is provided by the implementation of a method for determining the quality of cell culture, including mass spectrometric analysis of surface cell proteins.
  • a method for determining the quality of cell culture including mass spectrometric analysis of surface cell proteins.
  • 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, the presence of qualitative characteristics of the studied cell culture is monitored by comparing the totality of the obtained masses with the totality of spectrometrically determined masses of fragments of surface proteins of living cells with known qualitative characteristics.
  • trypsin is used as the protease.
  • trypsin is used as the protease.
  • the presence of quality characteristics that determine the suitability for antitumor immunotherapy is monitored.
  • the cells are monitored for the presence of qualitative characteristics indicating the absence of changes in the phenotype of cells of the studied cell culture during cultivation.
  • a primary cell culture can be used.
  • the cleaved fragments of surface proteins can be deglycosylated before mass spectrometric analysis.
  • FIG. 1 is a diagram of the preparation of a proteomic footprint of cultured cells according to the present invention.
  • FIG. 2 proteomic footprints of fibroblasts from various human tissues, classified by hierarchical cluster analysis.
  • FIG. 3 is a mass spectrum of surface protein fragments and a corresponding footprint obtained according to the second embodiment of the invention from a freshly initiated primary culture of human tumor cells (3A); the mass spectrum of surface protein fragments and the corresponding footprint obtained from the same cell culture after a short cultivation (ST); the mass spectrum and the corresponding footprint obtained from the same cell culture after a longer cell culture (3B).
  • fibroblasts are almost identical (have a fusiform shape).
  • fibroblasts obtained from various sources differ significantly in their properties, and accordingly, have different indications for use in cell therapy.
  • fibroblasts derived from human adipose tissue are pluripatent similar to stem cells (see, for example, Zuk P.A. et al, "Numap adiroce tens ise source of multitett sells", MoI. Biol. CeIl., 2002, v. 13, 4279-4295).
  • Fibroblasts of human skin have a weak potential for reproduction, but are suitable for mesotherapy with large doses of multiplied cells of the patient himself.
  • Embryonic fibroblasts of human skin have good potential for reproduction and are suitable for allogeneic cell transplantation (Sukhikh G.T., “Fetal cell transplantation in medicine: present and future”, Bull. Exp. Biol. Med., 1998, 126, Appl. , 3-13).
  • Dermal papilla fibroblasts hair follicle possess trichogenic properties and can be used in cell therapy of alopecia (see, for example, the patent "Biotransplant, method for its preparation (options) and method for the treatment of alopecia", application: 2004125092/15, publ. 18.08.2004).
  • adipogenic, fetal fibroblasts and fibroblasts of the dermal papilla of the hair follicle were obtained from appropriate sources according to the previously described methods.
  • Primary skin fibroblast cultures were obtained according to the Rittié and Fishér methods (“Isolatiop apulture of ski ski fibroblasts”, Methods MoI. Med. 2005, v. 117, 83-98).
  • Primary cultures of adipogenic fibroblasts were obtained according to the methodology of Zuk et al.
  • a cell preparation containing the tested fibroblasts is poured into a culture bottle under sterile conditions. Fill growth vial medium (DMEM with 10% fetal bovine serum) and incubated at 37 ° C in
  • a 0.0001% trypsin solution is added to the cells using 1 ml of a solution per 25 cm 2 of the surface of the culture vial.
  • a trypsin solution taken from the cells is used for mass spectrometric determination of the aggregate mass of fragments of surface proteins corresponding to the analyzed cells.
  • the measured set of masses of fragments of surface proteins of the analyzed cells is compared with the set of masses of fragments of surface proteins of cell cultures obtained in a similar way with known qualitative characteristics.
  • Mass spectrometric analysis is carried out as follows. Samples obtained during the incubation of cells with trypsin (see paragraph N ° 4 of the protocol) are desalted using tips for automatic pipettes with reversed phase ZipTipc 18 (Milliroe Corp., USA), in accordance with the manufacturer's protocol. The masses of peptide fragments contained in the sample are measured by time-of-flight (MALDI-TOF) mass spectrometry. 2 ⁇ l of the desalted solution is mixed on a mass spectrometric target in a 1: 1 ratio with a saturated solution of ⁇ -cyano-4-hydroxy-hydroxyquinamine acid containing 50% acetonitrile and 0.5% trifluoroacetic acid.
  • MALDI-TOF time-of-flight
  • the sample droplets obtained on the target are dried in air and mass spectrometric analysis is carried out in the mass range of peptides from 600 to 4000 Da.
  • the resulting mass spectra corresponding to different cell lines are pre-processed for subsequent classification by hierarchical cluster analysis.
  • the list of masses of peptides is translated into binary code ("1" - is a certain mass of the peptide in the spectrum and, accordingly, “O” means the absence of the mass of the peptide), thus generating a proteomic footprint of the cell culture.
  • the classification (grouping) of footprints is carried out by clustering according to the Ward method, using the squares of Euclidean distances to calculate the distance matrix.
  • FIG. Figure 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).
  • having a test cell preparation of fibroblasts it is possible to accurately determine its origin by the fact that its footprint belongs to one or another cluster. For example, if the test cell preparation is matched with an asterisk (*) in FIG.
  • the so-called proteomic footprint is a kind of "barcode” characterizing cells for their relation to fibroblasts with certain properties of interest for cell therapy.
  • the protease treatment conditions of the cells are determined experimentally for each cell type and the activity of the used protease, and can vary significantly from 0.00001% to 0.05%, for the protease concentration, and from 30 seconds to 10 minutes for the processing time of the cells. In the case of using trypsin with a different activity, its concentration changes in direct proportion to an increase or decrease in activity.
  • any other mass spectrometry option is used.
  • Trypsin solution can be prepared both in sterile physiological saline and in any suitable saline or buffer solution.
  • proteases can be used, for example, chymotrypsin, proteinase K, etc.
  • the cleaved fragments of surface proteins can be deglycosylated, for example, pronase, before mass spectrometric analysis.
  • proteases instead of trypsin, a combination of proteases can be used.
  • cells In the case of using a cell suspension culture, cells must be sedimented to the bottom before sampling, for example by centrifugation, or any other suitable method may be used to remove cells from the test sample.
  • Desalting and concentration of the sample peptides for mass spectrometric analysis can be carried out by any suitable method. In other embodiments of the invention, if the applied mass spectrometric analysis protocol does not require desalination and / or concentration of the analyzed sample containing fragments of surface proteins, no desalination and / or concentration is carried out.
  • any suitable mathematical processing of the measured masses of protein fragments is used, which allows one to correctly classify cellular material according to the properties of interest, for example, by applying regression analysis, K-mes clustering, neural networks, PCA, SVM, SOM, etc., as well as their combinations.
  • the patented method can be applied to any type of cell preparation, in particular to a suspension of cells, mixed cultures, organotypic cultures and cell aggregates (granules, spheroids), as well as to freshly isolated cells and tissue fragments.
  • the invention is further illustrated by a second example of assessing the quality of a culture of human colon cancer tumor cells for their suitability for antitumor vaccination.
  • Cells isolated from a patient’s tumor, cultured and subsequently used for antitumor vaccination should be identical to the patient’s tumor cells.
  • cell cultivation significantly distorts the phenotype of the primary culture due to the impossibility of artificially reconstructing vitro conditions under which tumor cells grew in the patient's body.
  • the quality of cultured tumor cells used for vaccination is subject to mandatory control and is expressed in the degree to which they correspond to the patient’s tumor cells. A protocol for establishing such compliance is presented below.
  • the growth medium is removed from the vial with cultured tumor cells and the cells are washed three times with 0.9% sodium chloride solution or silane phosphate buffer, each time using a volume equal to at least half the volume of the growth medium poured into the culture vial. As a result of cell washing, traces of serum contained in the growth medium should be removed. 2. A 0.0001% trypsin solution (activity -3000 U / mg) is added to the cells using 1 ml of solution per 25 cm 2 of the surface of the culture vial.
  • Mass spectrometric analysis is carried out according to the protocol of mass spectrometry of glycosylated peptides (Tajiri M. et al., "Diffépal apulusis on situs on on paculum fibropesto: .15, 1332-1340).
  • 140 ⁇ l of the analyzed solution of surface protein fragments are mixed with 140 ⁇ l of ethanol, 720 ⁇ l of butanol and 15 ⁇ l of CL4B Sepharose are added. Incubate with slow stirring for 45 minutes. After incubation, Sepharose is washed twice with a solution with the same alcohol and butanol content and incubated for 30 min in a 50% ethanol solution. The ethanol solution was removed from sepharose and dried on a rotary evaporator. The resulting precipitate was dissolved in 10 ⁇ l of water and analyzed by time-of-flight (MALDI-TOF) mass spectrometry.
  • MALDI-TOF time-of-flight
  • 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 sample droplets obtained on the target are dried in air and mass spectrometric analysis is carried out in the peptide mass range from 1000 to 4000 Da.
  • a repeated (control) mass spectrometric analysis of cell culture reproduces at least 95% of the mass of protein fragments, which can be considered a criterion for the identity of the two compared cell preparations. It is proposed to evaluate the suitability of a cell preparation for vaccinating a 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 human colon cancer cells.
  • FIG. ZB 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 initial spectrum obtained from freshly isolated tumor cells.
  • the pollutant presents a mass spectrum of the same cells propagated by cultivation (2nd passage), but no longer suitable for antitumor vaccination. During cultivation, significant changes took place in the surface proteins of the cells (see, for example, the 1800-2000 Da footprint region in FIG. 3B), which allowed them to be considered non-identical to the cells of the original donor tumor.
  • barcode i.e. the indicator that most objectively characterizes cells.
  • control of the presence of qualitative characteristics of the studied cell culture by comparing the totality of the obtained masses of fragments of the surface proteins of its cells with the combination of mass spectrometrically determined masses of fragments of the surface proteins of living cells with known qualitative characteristics makes it possible to most accurately select high-quality (with expected useful properties) cells.
  • the obtained mass spectra can be transformed with the possibility of numerical expression of the analysis results, which is necessary to implement the GMP 5 standard, which excludes the human factor in assessing the quality of cell culture, and will ensure the necessary objectivity of the results.
  • the accuracy of determining the presence of qualitative characteristics of the studied cell culture during the implementation of the patented method is also ensured by the use of living cells of the studied cell culture, which are subjected to vital proteases. Cells during processing by vital exposure to proteases do not die, which makes it possible to avoid contamination of the analyzed samples with major proteins of the cell cytoplasm and significantly reduce the consumption of cellular material in the analysis of cell culture.
  • Pre-washing of cells which is necessary to remove traces of serum contained in the growth medium, also avoids contamination of the analyzed samples.
  • the patented method allows to characterize the cells according to the most relevant, including for cell transplantology, surface proteins.

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Abstract

L'invention concerne des technologies cellulaires et notamment l'analyse de cellules cultivées in vitro et peut s'utiliser en biotechnologie, en cosmétologie, en médecine (transplantologie cellulaire), dans la fabrication de matériau cellulaire caractérisé possédant des propriétés curatives ou cosmétologiques. Le procédé de détermination de la qualité d'une culture cellulaire comprend une analyse spectrométrique de masse des protéines cellulaires superficielles. On soumet des cellules vivantes préalablement nettoyées de la culture cellulaire étudiée à l'action vitale de la protéase, on sélectionne des fragments clivés de protéines superficielles et on détermine leur masse par spectrométrie de masse. On contrôle la présence de caractéristiques qualitatives dans la culture cellulaire examinée par la comparaison de la totalité des masses obtenues avec l'ensemble de fragments de masses déterminées par spectrométrie de masse des protéines superficielles de cellules vivantes ayant des caractéristiques qualitatives connues. L'invention permet d'améliorer l'objectivité (la précision) d'identification de cellules cultivées in vitro à des fins de sélection des cellules qualitatives (possédant des propriétés utiles attendues) et à réduire les dépenses matérielles et le temps requis.
PCT/RU2007/000571 2007-04-06 2007-10-16 Procédé pour tester la qualité d'une culture de cellules Ceased WO2008123793A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12366580B2 (en) 2018-09-11 2025-07-22 Juno Therapeutics, Inc. Methods for mass spectrometry analysis of engineered cell compositions
US12379375B2 (en) 2017-04-14 2025-08-05 Juno Therapeutics, Inc. Methods for assessing cell surface glycosylation

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2670001C1 (ru) * 2017-12-25 2018-10-17 Федеральное государственное бюджетное учреждение науки Институт биохимии и физиологии растений и микроорганизмов Российской академии наук Способ получения белков клеточной поверхности

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SU944580A1 (ru) * 1980-02-07 1982-07-23 Кировский научно-исследовательский институт переливания крови Способ получени поверхностного антигена гепатита В
WO2003025565A2 (fr) * 2001-09-21 2003-03-27 Caprion Pharmaceuticals Inc. Preparation de membranes plasmiques fortement purifiees
US6861234B1 (en) * 2000-04-28 2005-03-01 Mannkind Corporation Method of epitope discovery
KR20060015604A (ko) * 2003-05-13 2006-02-17 모노퀀트 피티와이 리미티드 Ｔ-세포 수용체 ｖ/ｄ/ｊ 유전자 내의 반복 서열에 의한클론 세포의 확인 방법
WO2006051405A2 (fr) * 2004-11-12 2006-05-18 Cambridge University Technical Services Ltd. Procedes et moyens lies aux cellules souches cancereuses

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
PT1000154E (pt) * 1997-07-23 2007-03-30 Roche Diagnostics Gmbh Identificação de linhas celulares humanas para a produção de proteínas humanas por activação endógena de genes
JP2001249125A (ja) * 2000-03-06 2001-09-14 Suntory Ltd 組織または細胞における物質分析のための方法
US7164552B2 (en) * 2003-09-29 2007-01-16 Hitachi Global Storage Technologies Netherlands B.V. Method for self-servo writing a disk drive with dual-stage actuator

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SU944580A1 (ru) * 1980-02-07 1982-07-23 Кировский научно-исследовательский институт переливания крови Способ получени поверхностного антигена гепатита В
US6861234B1 (en) * 2000-04-28 2005-03-01 Mannkind Corporation Method of epitope discovery
WO2003025565A2 (fr) * 2001-09-21 2003-03-27 Caprion Pharmaceuticals Inc. Preparation de membranes plasmiques fortement purifiees
KR20060015604A (ko) * 2003-05-13 2006-02-17 모노퀀트 피티와이 리미티드 Ｔ-세포 수용체 ｖ/ｄ/ｊ 유전자 내의 반복 서열에 의한클론 세포의 확인 방법
WO2006051405A2 (fr) * 2004-11-12 2006-05-18 Cambridge University Technical Services Ltd. Procedes et moyens lies aux cellules souches cancereuses

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12379375B2 (en) 2017-04-14 2025-08-05 Juno Therapeutics, Inc. Methods for assessing cell surface glycosylation
US12366580B2 (en) 2018-09-11 2025-07-22 Juno Therapeutics, Inc. Methods for mass spectrometry analysis of engineered cell compositions

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