WO2012107447A2 - Milieu de culture et procédé pour l'extraction, l'isolement et la propagation de lignées cellulaires animales de haute pureté et lignées cellulaires animales continues hautement purifiées ainsi obtenues - Google Patents

Milieu de culture et procédé pour l'extraction, l'isolement et la propagation de lignées cellulaires animales de haute pureté et lignées cellulaires animales continues hautement purifiées ainsi obtenues Download PDF

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WO2012107447A2
WO2012107447A2 PCT/EP2012/052054 EP2012052054W WO2012107447A2 WO 2012107447 A2 WO2012107447 A2 WO 2012107447A2 EP 2012052054 W EP2012052054 W EP 2012052054W WO 2012107447 A2 WO2012107447 A2 WO 2012107447A2
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culture medium
cell lines
vascular
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Gloria INVERNICI
Silvia CRISTINI
Stefania Elena NAVONE
Eugenio Agostino PARATI
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Fondazione I.R.C.C.S. Istituto Neurologico "Carlo Besta"
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
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    • C12N2501/91—Heparin

Definitions

  • the present invention relates to a culture medium for the extraction, isolation and propagation of animal cell lines of high purity deriving from vascular and/or endothelial structures of cardiocirculatory and/or mesodermic systems and to a method for extracting, isolating and propagating animal cell lines and continuous animal cell lines of high purity thus obtained.
  • Endothelial tissue is a particular type of epithelial tissue, of mesenchymal derivation, morphologically similar to simple squamous epithelial tissue, which lines the interior surface of blood vessels and the heart (Scott PAE et al. 1993).
  • the endothelium comes into contact with a large number of blood molecules and its function is to act as an interface and respond, with the release of suitable mediators, to the stimuli received in order to maintain vascular homeostasis.
  • vascular atherosclerosis vascular atherosclerosis
  • arterial hypertension hypertension
  • heart failure the endocardial endothelium is involved in modulating left ventricular function
  • diabetes acute and chronic hyperglycaemia plays an important role in endothelial dysfunction and leads to vascular complications in diabetic disease: micro- and macroangiopathy
  • erectile dysfunction multiple sclerosis
  • endometriosis AIDS
  • bacterial infections autoimmune diseases, such as lupus erythematosus.
  • endothelial activation corresponds to a response that is different from that of a healthy endothelium, if not completely the opposite (Nagashima T et al. 1999).
  • Endothelial cells act as a selective barrier to the passage of molecules and cells between blood and organs: permeability depends on specific protein channels on the membrane thereof. This is particularly true at the cerebral level, where the endothelium forms the so-called "blood-brain barrier”: a veritable barrier that is highly selective.
  • the BBB is an anatomo-functional unit resulting from the particular characteristics of the endothelial cells which compose the vessels of the Central Nervous System (CNS) and mainly has a function of protecting brain tissue from harmful elements (e.g. chemical) present in the blood while nonetheless permitting the passage of substances necessary for metabolic functions.
  • CNS Central Nervous System
  • the endothelial cell is also a subject of study in the oncological realm.
  • One of the most innovative aspects of antitumour therapies is the possibility of inhibiting tumour growth by blocking the supply of blood. If "starved", the tumour will not grow but on the contrary it will shrink and become more vulnerable to chemotherapy and radiotherapy.
  • the current techniques used to isolate and expand animal vascular and/or endothelial cells consist in immunoselection or selection based on the different abilities to adhere to a substrate (pre-plating), the aim being to purify said vascular and/or endothelial animal cells and eliminate the contaminated populations that impair the purity and proliferation thereof.
  • the present solutions used allow only a limited expansion of said vascular and/or endothelial animal cells deriving from cardiocirculatory and/or mesodermic systems, since the vascular and/or animal cells are susceptible to senescence and the occurrence of possible contaminants.
  • the vascular and/or endothelial cells isolated from human and mouse cardiocirculatory and/or mesodermic systems show scant proliferative potential and any contaminating cells present will prevail over the vascular and/or endothelial cells, causing the vascular and/or endothelial colonies to die due to suffocation and deprival of growth factors after a few days of culture (about a week).
  • vascular and/or endothelial cells involves the creation of "immortalized” cell lines. This has made it possible to obtain large numbers of endothelial cells which are genetically manipulated and, as such, artificial organisms that depart from the natural starting conditions, because cellular aging is a process at the basis of all physiological phenomena.
  • Cell immortalization is obtained by introducing oncogenes, which, by altering the cellular cycle of the cell, prevent it from undergoing a normal biological course, with a higher likelihood of transforming into a tumour cell (not coincidentally, the first immortalized cell line was isolated by George Gay in 1951 from cervical tumour tissue (HeLa)).
  • the technical task that the present invention has set itself is to provide a culture medium and a method for the extraction, isolation and propagation of animal cell lines deriving from vascular and/or endothelial structures of cardiocirculatory and/or mesodermic systems which is capable of obtaining unalterable, continuous, non-engineered cells lines of high purity, with a high proliferative potential and high survival rate.
  • a culture medium for the extraction, isolation and propagation of animal cell lines of high purity deriving from vascular and/or endothelial structures of cardiocirculatory and/or mesodermic systems comprising glucides, inorganic salts, vitamins, amino acids essential for protein synthesis, including at least glutamine, haemoderivatives or the like for the contribution of at least metalloproteins, hormones and trophic factors, present in variable proportions so as to assure a trophic equilibrium for cell proliferation, and having a pH in a range of between 6.8 and 7.4, characterised in that said culture medium has a formulation comprising:
  • HEPES in an interval between 1 and 1000 ⁇ g/mL
  • Epidermal growth factor in an interval between 1 and 200 ng/mL
  • Fibroblast growth factor in an interval between 1 and 150 ng/mL
  • Hydrocortisone (HC) in an interval between 0.1 and 50
  • Bovine serum albumin in an interval between 1 and 200 ⁇
  • the culture medium further comprises bovine brain extract (BBE) in an interval between 1 and 100 ⁇ g/mL.
  • BBE bovine brain extract
  • the ratio of the concentrations present in the culture medium is 1 : 10 between amino acids and glucides, 1 :4.5 between hormones and trophic factors and 1 : 13 between metalloproteins and mineral salts.
  • the haemoderivatives or the like present in the culture medium comprise serum in a concentration of 2.5%.
  • the invention discloses a culture medium for the extraction, isolation and propagation of animal cell lines of high purity deriving from vascular and/or endothelial structures of cardiocirculatory and/or mesodermic systems, comprising:
  • EGF Epidermal growth factor
  • bFGF Basic fibroblast growth factor
  • BSA Bovine serum albumin
  • the culture medium further comprises bovine brain extract (BBE) at a concentration of 6 ⁇ g/mL.
  • BBE bovine brain extract
  • the present invention further discloses a method of extracting, isolating and propagating animal cell lines of high purity deriving from vascular and/or endothelial structures of cardiocirculatory and/or mesodermic systems, comprising a first step of plating a cell suspension deriving from said vascular and/or endothelial structures with the culture medium of the invention, and a step of progressive purification and proliferation of the cell line by reiteration of a cycle of aspiration of the culture medium in order to remove the non-adhering component of said cell suspension, and replenishment with fresh culture medium to replace the aspirated medium.
  • the method of extracting, isolating and propagating of animal cell lines of high purity deriving from vascular and/or endothelial structures of cardiocirculatory and/or mesodermic systems comprises at least a step of centrifuging the removed culture medium in order to separate the non-adhering component of the cell suspension, and a subsequent step of re-plating said component with fresh culture medium in order to create a new cell line.
  • the method of extracting, isolating and propagating of animal cell lines of high purity comprises a step of cryoconservation of the cell lines obtained.
  • the present invention discloses continuous animal cell lines of high purity, particularly human or rodent cell lines, deriving from vascular and/or endothelial structures of cardiocirculatory and/or mesodermic systems obtained with the above-described method of extraction, isolation and propagation.
  • the animal cell lines have a purity of no less than 96%.
  • Figure 1 shows the characterisation of endothelial cells (ECs) isolated from adult human brain tissue.
  • the images show the typical endothelial morphology of endothelial cells derived from an adult human brain as observed under a microscope (magnification 50X) and the ability thereof to form tubes
  • the figure confirms the endothelial character, evaluated on the basis of the phenotypic expression of CD analysed by FACS and the expression of typical endothelial markers (e.g.: CD31) as well as functional tests such as the expression of e-NOS and Glut-1 (magnification 200X).
  • typical endothelial markers e.g.: CD31
  • functional tests such as the expression of e-NOS and Glut-1 (magnification 200X).
  • Figure 2 shows the characterisation of endothelial cells isolated from foetal human brain tissue.
  • the images show the typical endothelial morphology of ECs derived from foetal human brain tissue as observed under a microscope (magnification 100X) and the ability thereof to form tubes (magnification 100X).
  • the figure confirms the endothelial character, evaluated on the basis of the phenotypic expression of CD analysed by FACS and the expression of typical endothelial markers (e.g.: CD31) as well as functional tests such as the expression of e-NOS and Glut-1 (magnification 200X).
  • Figure 3 shows the characterisation of endothelial cells isolated from adult CD1 mouse brain tissue.
  • the images show the typical endothelial morphology of ECs derived from an adult brain of the CD1 mouse strain as observed under a microscope (magnification 50X) and the ability thereof to form tubes
  • the figure confirms the endothelial character, evaluated on the basis of the phenotypic expression of CD analysed by FACS and the expression of typical endothelial markers (e.g.: CD31) as well as functional tests such as the expression of e-NOS and Glut-1 (magnification 200X).
  • typical endothelial markers e.g.: CD31
  • functional tests such as the expression of e-NOS and Glut-1 (magnification 200X).
  • Figure 4 shows the characterisation of endothelial cells derived from adult wr/wr mouse brain tissue.
  • the images show the typical endothelial morphology of endothelial cells derived from an adult brain of the wr/wr mouse strain as observed under a microscope (magnification 50X) and the ability thereof to form tubes
  • the figure confirms the endothelial character, evaluated on the basis of the phenotypic expression of CD analysed by FACS and the expression of typical endothelial markers (e.g.: CD31) as well as functional tests such as the expression of e-NOS and Glut-1 (magnification 200X).
  • typical endothelial markers e.g.: CD31
  • functional tests such as the expression of e-NOS and Glut-1 (magnification 200X).
  • Figure 5 shows the characterisation of endothelial cells derived from adult
  • NOD/SCID mouse brain tissue The images show the typical endothelial morphology of endothelial cells derived from an adult brain of the NOD/SCID mouse strain as observed under a microscope (magnification 50X) and the ability thereof to form tubes (magnification 100X). The figure confirms the endothelial character, evaluated on the basis of the phenotypic expression of CD analysed by
  • Figure 6 shows the characterisation of endothelial cells derived from adult CD1 mouse heart tissue.
  • the images show the typical endothelial morphology of endothelial cells derived from adult heart tissue of the CD1 mouse strain as observed under a microscope (magnification 50X) and the ability thereof to form tubes (magnification 100X).
  • the figure confirms the endothelial character, evaluated on the basis of the phenotypic expression of CD analysed by FACS and the expression of typical endothelial markers (e.g.: CD31) as well as functional tests such as the expression of e-NOS (magnification 200X).
  • FIG. 7 shows the characterisation of human microvascular endothelial cells
  • HMVECs The images show the typical endothelial morphology of HMVECs observed under a microscope (magnification 50X) and the ability thereof to form cords (magnification 100X). The figure confirms the endothelial character, evaluated on the basis of the phenotypic expression of CD analysed by FACS and the expression of typical endothelial markers (e.g.: CD31) as well as functional tests such as the expression of e-NOS (magnification 200X).
  • typical endothelial markers e.g.: CD31
  • functional tests such as the expression of e-NOS (magnification 200X).
  • Figures 8, 9 A, 9B, 10 represent micrographs showing comparative examples of the isolation, expansion and proliferation of human and mouse microvascular endothelial cells using a prior art formulation, a commercial endothelial standard medium (CEM) and the medium of the invention EndoPM.
  • the culture medium disclosed by the present invention comprises a combination of different essential elements and several supplementary elements for isolating, purifying and propagating endothelial and/or vascular cells from healthy and non- healthy, human and mouse brain and non-brain tissues, without any need for immunoselection or genetic engineering.
  • the vascular and/or endothelial cells thus obtained are characterised by high purity, a high proliferative potential and cryoconservability and are able to maintain their characteristics without alteration during the culture steps.
  • One formulation of the medium in accordance with the present invention envisages using as starting point, in general, any known serum-free basal medium at a concentration of IX, such as, for example, DMEM/F12.
  • the basal medium is combined with haemoderivatives that represent a source of protein and growth factors in general, essential for cell proliferation, in particular serum at a concentration of 2.5%.
  • EGF Epidermal growth factor
  • bFGF Basic fibroblast growth factor
  • the formulation of the culture medium further includes the following complementary elements:
  • BSA Bovine serum albumin
  • BBE Bovine brain extract
  • HEPES (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid) at a concentration of 596 - Heparin at a concentration of 2 ⁇ g/mL
  • EGF Epidermal growth factor
  • bFGF Basic fibroblast growth factor
  • the medium thus formulated provides optimal culture conditions for eliminating non-endothelial (contaminating) cells in the initial phases and simultaneously creating a situation conducive to the proliferation of vascular and/or endothelial cells.
  • the pH of the medium is buffered to a pH in the range of 6.8 to 7.4 using sodium bicarbonate.
  • the presence of an indicator such as phenol red will allow the pH of the culture medium to be monitored.
  • a yellowish colour indicates an acidic pH value, whereas purplish red corresponds to an alkaline one.
  • An innovative feature is the possibility of isolating from brain tissue and easily expanding colonies of endothelial cells of high purity and in large number for the purpose of studying the mechanisms involved in the physiopathology of numerous cerebral and cerebrovascular pathologies and in tumour processes.
  • a change in endothelial tissue is in fact the first manifestation of a pathological event.
  • the importance of this invention implies the possibility of monitoring over time the evolution of the endothelial response to different mechanical and pharmacological stimuli in cultures of healthy and pathological brain endothelial cells of human and mouse origin.
  • the isolation method includes a number of steps.
  • the tissue from which the vascular structures are to be isolated is reduced to pieces with the aid of a sterile knife and then digested with the enzyme Roche Liberase Blendzyme II at a concentration of 2.5 mg/ml until complete digestion of the tissue.
  • the cell suspension is plated on a flask/well - previously collagen coated (bovine collagen of the type produced by BD Biosciences) - with the culture medium of the present invention.
  • the medium is gently aspirated and fresh medium of the present invention is added.
  • the non-adhering cell populations are centrifuged and seeded in a new collagen-coated flask/well with the medium of the present invention.
  • the same technique is applied after 7 days and every 7 days a medium change takes place.
  • the endothelial cells thus obtained are cultured for at least 30 days until endothelial colonies have formed and the "contaminating" part has been reduced.
  • the cells are detached with TrypLETM Select (Invitrogen).
  • the culture medium thus formulated can be used for the purpose of studying rolling and homing aspects, developing cell therapies and studying the district-specific features of endothelial cells, thus enabling the development of target therapies and of specific diagnostic markers.
  • the realms of application of the present invention include Cellular Biology Laboratories, Regenerative Medicine Laboratories, organ transplants, in vitro study of the toxicity of marketed drugs, in vivo and in vitro study of human pathologies and in animal models for the purpose of analysing the role of endothelial cells in the origin and development of disease.
  • the brain tissue was obtained from cadavers and from patients who underwent surgical treatment for brain trauma and gave their informed consent.
  • the brain tissue was reduced to pieces with the aid of a sterile knife and then digested with the enzyme Roche Liberase Blendzyme II at a concentration of 2.5 mg/ml until complete digestion of the tissue.
  • the cell suspension was plated on a previously collagen-coated (bovine collagen of the type produced by BD Biosciences) flask/well containing the medium of the present invention.
  • the endothelial cells thus obtained were cultured for at least 30 days until endothelial colonies were formed and the "contaminating" part was reduced.
  • Fig 1 The endothelial morphology of the cells was examined using a Zeiss inverted optical microscope (Fig 1).
  • the cultured cells were identified as endothelial cells using a variety of methods, namely, FACS analysis, immunocytochemistry and the cord formation test. Functional tests were performed for GLUT-1 and e-NOS expression (Fig 1) (Pardridge WM. et al. 1990; Marsden PA. et al. 1992).
  • the brain tissue was obtained from foetal tissue in accordance with law 194, subject to the mother's informed consent and based on an agreement between the Foundation IRCCS of the Neurological Institute "C. Besta” and the Foundation IRCCS of the Mangiagalli Regina Maria Policlinic.
  • the brain tissue was reduced to pieces with the aid of a sterile knife and then digested with the enzyme Roche Liberase Blendzyme II at a concentration of 2.5 mg/ml until complete digestion of the tissue.
  • the cell suspension was plated on a previously collagen-coated (bovine collagen of the type produced by BD Biosciences) flask/well containing the medium of the present invention.
  • Fig 2 When the cell population reached confluence, the cells were detached with TrypLETM Select (Invitrogen). The endothelial morphology of the cells was examined using a Zeiss inverted optical microscope (Fig 2). The cultured cells were identified as endothelial cells using a variety of methods, namely, FACS analysis, immunocytochemistry and the cord formation test. Functional tests were performed for GLUT-1 and e-NOS expression (Fig 2).
  • the brain tissue was drawn from the CD1 mouse strain.
  • the brain isolated from 3 animals was reduced to pieces with the aid of a sterile knife and subsequently digested with the enzyme Roche Liberase Blendzyme II at a concentration of 2.5 mg/ml until complete digestion of the tissue.
  • the cell suspension was plated on a previously collagen-coated (bovine collagen of the type produced by BD Biosciences) flask/well containing the medium of the present invention.
  • the endothelial cells thus obtained were cultured for at least 30 days until endothelial colonies formed and the "contaminating" part was reduced.
  • the endothelial morphology of the cells was examined using a Zeiss inverted optical microscope (Fig 3).
  • the cultured cells were identified as endothelial cells using a variety of methods, namely, FACS analysis, immunofluorescence and the cord formation test. Functional tests were performed for GLUT-1 and e-NOS expression (Fig 3).
  • the endothelial cells thus obtained were cultured for at least 30 days until endothelial colonies formed and the "contaminating" part was reduced.
  • the endothelial morphology of the cells was examined using a Zeiss inverted optical microscope (Fig 4).
  • the cultured cells were identified as endothelial cells using a variety of methods, namely, FACS analysis, immunofluorescence and the cord formation test. Functional tests were performed for GLUT-1 and e-NOS expression (Fig 4).
  • EXAMPLE 5 Isolation and expansion of endothelial cells from adult NOD/SCID mouse brain tissue.
  • the brain tissue was drawn from the NOD/SCID mouse strain.
  • the brain isolated from 3 animals was reduced to pieces with the aid of a sterile knife and then digested with the enzyme Roche Liberase Blendzyme II at a concentration of 2.5 mg/ml until complete digestion of the tissue.
  • the cell suspension was plated on a previously collagen-coated (bovine collagen of the type produced by BD Biosciences) flask/well containing the medium of the present invention.
  • the endothelial cells thus obtained were cultured for at least 30 days until endothelial colonies formed and the "contaminating" part was reduced.
  • the endothelial morphology of the cells was examined using a Zeiss inverted optical microscope (Fig 5).
  • the cultured cells were identified as endothelial cells using a variety of methods, namely, FACS analysis, immunofluorescence and the cord formation test. Functional tests were performed for GLUT-1 and e-NOS expression (Fig 5).
  • the heart tissue was drawn from the CD1 mouse strain.
  • the heart isolated from 3 animals was reduced to pieces with the aid of a sterile knife and then digested with the enzyme Roche Liberase Blendzyme II at a concentration of 2.5 mg/ml until complete digestion of the tissue.
  • the cell suspension was plated on a previously collagen-coated (bovine collagen of the type produced by BD Biosciences) flask/well containing the medium of the present invention.
  • the endothelial cells thus obtained were cultured for at least 30 days until endothelial colonies formed and the "contaminating" part was reduced.
  • the endothelial morphology of the cells was examined using a Zeiss inverted optical microscope (Fig 6).
  • the cultured cells were identified as endothelial cells using a variety of methods, namely, FACS analysis, immunofluorescence and the cord formation test.
  • HMVECs human microvascular endothelial cells
  • the cell line used in the present study as the control line is commercially available.
  • the cells were cultured in EBM medium (Endocult Basal Medium, Lonza Group).
  • Figures 8, 9 A, 9B and 10 represent micrographs showing comparative examples of the isolation, expansion and proliferation of human and mouse microvascular endothelial cells using a prior art formulation, a commercial endothelial standard medium (CEM) and the medium of the invention EndoPM (Fig. 8, 9A, 9B, 10).
  • CEM commercial endothelial standard medium
  • EndoPM the medium of the invention EndoPM
  • the proliferation index represents the percentage of the cell number at a specific time point (n) divided by the number of input cells at time 0
  • the survival index represents the percentage of the number of harvested cells 12 hours after plating into EndoPM and CEM.
  • EndoPM facilitate the mitosis in endothelial cells enhancing the time of duplication; in fact, after appropriate synchronization time, starved cells were treated with complete EndoPM or CEM medium for 5h or overnight and in all the samples examined the cells in mitosis were more when treated with EndoPM.

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  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne un milieu de culture pour l'isolement et la propagation de lignées cellulaires animales de pureté élevée issues de structures vasculaires et/ou endothéliales des systèmes cardio-circulatoire et/ou mésodermique, comprenant des glucides, des sels inorganiques, des vitamines, des acides aminés essentiels pour la synthèse protéique, comprenant au moins de la glutamine, des hémodérivés ou similaires, pour la contribution d'au moins des métalloprotéines, des hormones et des facteurs trophiques, présents dans des proportions variables afin d'assurer un équilibre trophique pour la prolifération cellulaire, et ayant un pH se situant dans la plage entre 6,8 et 7,4.
PCT/EP2012/052054 2011-02-11 2012-02-07 Milieu de culture et procédé pour l'extraction, l'isolement et la propagation de lignées cellulaires animales de haute pureté et lignées cellulaires animales continues hautement purifiées ainsi obtenues WO2012107447A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP12702829.8A EP2673356A2 (fr) 2011-02-11 2012-02-07 Milieu de culture et procédé pour l'extraction, l'isolement et la propagation de lignées cellulaires animales de haute pureté et lignées cellulaires animales continues hautement purifiées ainsi obtenues

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ITMI2011A000201 2011-02-11
ITMI2011A000201A IT1408248B1 (it) 2011-02-11 2011-02-11 Terreno di coltura e metodo per l estrazione, l isolamento e la propagazione di linee cellulari endoteliali animali ad elevata purezza e linee cellulari animali continue altamente purificate così ottenute

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WO2012107447A2 true WO2012107447A2 (fr) 2012-08-16
WO2012107447A3 WO2012107447A3 (fr) 2012-11-29

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PCT/EP2012/052054 WO2012107447A2 (fr) 2011-02-11 2012-02-07 Milieu de culture et procédé pour l'extraction, l'isolement et la propagation de lignées cellulaires animales de haute pureté et lignées cellulaires animales continues hautement purifiées ainsi obtenues

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Country Link
EP (1) EP2673356A2 (fr)
IT (1) IT1408248B1 (fr)
WO (1) WO2012107447A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107043737A (zh) * 2017-06-20 2017-08-15 青岛金典生化器材有限公司 用于培养病毒的无血清培养基及其制备方法
GB2543374B (en) * 2015-05-08 2018-11-07 Imagen Therapeutics Ltd Personalised media

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
None

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2543374B (en) * 2015-05-08 2018-11-07 Imagen Therapeutics Ltd Personalised media
CN107043737A (zh) * 2017-06-20 2017-08-15 青岛金典生化器材有限公司 用于培养病毒的无血清培养基及其制备方法

Also Published As

Publication number Publication date
IT1408248B1 (it) 2014-06-13
ITMI20110201A1 (it) 2012-08-12
EP2673356A2 (fr) 2013-12-18
WO2012107447A3 (fr) 2012-11-29

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