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MAMMALIAN CELL CULTURE AND CO‐CULTURE MODELS
Maria A. DeliInstitute of Biophysics
„Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities”TÁMOP-4.1.1.C-13/1/KONV-2014-0001
• Introduction• Cell culture facility• Equipments• Cell culture ware• Sterilization• Culture media• Primary cultures• Cell lines, differentiation• Subculture (passage)• Freezing, storing and thawing cells• Contamination
INTRODUCTION TO MAMMALIAN TISSUE AND CELL CULTURE
BRIEF HISTORY OF TISSUE CULTURE
• 1907 Ross Harrison frog embryonic tissue• 1912 Alexis Carrel chick embryo heart• 1948 Katherine Sanford single cells• 1955 Harry Eagle culture medium• 1960s cell lines• 1979‐1981 different culture media• Recombinant protein expression and hybridoma
technique• Large scale culturing, industrial production
CELL CULTURE FACILITY
Floor plan for an ideal high‐use tissue culture facility
CELL CULTURE FACILITY
EQUIPMENTS FOR CELL CULTURECO2 INCUBATOR
Temperature 26‐39 ºC 37ºCGas concentration CO2 concentration 0‐10 % 5 %
O2 concentration 18‐1 %Humidity – water tray
EQUIPMENTS FOR CELL CULTUREPHASE CONTRAST MICROSCOPE
In‐phase out‐phase image of a cell culture
Phase plate and light annulus alignment
Position of objectives inverted
EQUIPMENTS FOR CELL CULTURELAMINAR FLOW (BIOHAZARD) CABINET
Biohazard cabinet: vertical air flow, HEPA filter, built‐in UV lampprotection of cells and researchers
EQUIPMENTS FOR CELL CULTURE
Desktop refrigerated centrifuge Vacuum pumpBottles: used medium
humidity trap
Cabinet for sterilized glassware and culture dishes
Pipet aids
EQUIPMENTS FOR CELL CULTURE
Glasswarepipets, tubes, Petri‐dishes, bottles, coverslips
Sterile disposable plasticwarepipetstubesdishesflasksmultiwell plates (6, 12, 24, 96‐well)cell culture insertsculture slides“Cellstack”“Cellmax” hollow fiber unitsfilter units
Surfacenon‐treated, TC‐treated, precoated
CELL CULTURE‐WARE
1. Hot air / Heat 3h at 160 °C or 1h at 180 °Cmetal, glass (eg. surgical instruments, glass pipettes)
2. Autoclaving 15‐30 min, 1 atm, 121 °Cliquids, bottles with cap, plasticware, papers, textiles
3. Gas sterilization ethylene oxide (hospitals), formaldehydeinstruments, catheters, big equipment, rooms
4. Irradiation 60 Co γ‐irradiationcommercial TC plasticware
5. Filtration 0.45 μm, 0.2 μm, 0.1 μm, different membranesassembling / disposablebuffers, protein solutions
Control of sterilization indicator tapesmicrobiological culture
STERILIZATION
CULTURE MEDIAHarry Eagle (1955) “..mixture of amino acids, vitamins, co‐factors, carbohydrates, and salts supplemented with a small
amount of serum protein..”
Basal Medium Eagle, Dulbecco’s Modified EagleMedium
• Provides nutrients and maintains chemical environment• Powdered or liquid • pH (6.5‐7.8, median: 7.2)
buffering systems; bicarbonate / CO2
H2O + CO2 = H2CO3 = H+ + CO3‐
zwitterionic buffers (Hepes) – Tris: toxic!phenol red
• Osmolarity (300 mOsm)salts and glucose
CULTURE MEDIA: COMPONENTS
• Main components
ions, glucose, amino acids, vitamins, co‐factors, trace elements, fatty acids
• Antibiotics• Serum
fetal bovine, fetal calf, calf, horse, etc.adhesion promoting componentsnutrients and trace mineralstransport proteins (transferrin, albumin)growth factors and hormonesstabilizing proteins (albumin, fetuin)
• Supplementsinsulin‐selenin‐transferrin
Cells taken from a tissue of a living organism and placed in culture (P0)After the first subculture: secondary culture (P1)After continued passages: cell line
Examples of primary cultures• White blood cells isolation from blood – lymphocytes, monocytes• Macrophage peritoneal lavage• Endothelial cells aorta, human coronary vessels, umbilical cord vein,
fat microvessels, brain microvessels• Epithelial cells kidney tubule, mammary, lung, colonic, choroid plexus• Nervous system cells glia, neurons• Muscle cells skeletal muscle, arterial smooth muscle• Other organs hepatocytes, pancreatic β‐cells, thymocytes• Skin fibroblasts, keratinocytes• Tumor cells human biopsies (carcinoma), rodent tumors
PRIMARY CULTURE
isolation of microvessel fragments
microvascular endothelial cellspuromycin treatment (Perriere et al., 2005)
Rat brain
astrocytes
Rat brain
PREPARATION OF PRIMARY CULTURES
Percoll gradient
MV fractionpericytes
mechanical & enzymic dissociation
Factor VIII‐related antigenPhase contrast microscopy
PRIMARY CULTUREcharacterization of brain capillary endothelial cells
ZO‐1 ß‐catenin Claudin‐5Walter F, Deli M
Fluorescent immunostaining
PRIMARY CULTUREAstroglia and brain capillary pericytes
GFAP
Phase contrast microscopy
Smooth muscle actin
Phase contrast microscopy
Veszelka et al. 2013
Established cell strains showing immortality
Spontaneous transformationin vivo – isolation from tumour tissue
HeLa human carcinoma cell linein vitro
Transformation of cells viral or oncogene transfection:
SV40, adenovirus vectors, polyoma virus
Tumour formation +/‐ in nude mice
Cell banks ATCC (American Type Culture Collection, USA)
LGC‐ATCC (UK)
CELL LINES 1.
CELL LINESIMMORTALIZED BRAIN ENDOTHELIAL CELLS
• GP8 (rat)Greenwood et al., 1996SV40 large T antigen
• RBE4 (rat)Durieu‐Trautmann et al., 1993Adenovirus‐vector (plasmid pE1A‐neo)
• D3 (human)Weksler et al., 2005Lentiviral hTERT + SV40 large T antigen
Deli M
CELL LINESPC12 rat phaeochromocytoma cells (adrenal gland)
Differentiated bynerve growth factor
Control
Deli M
CELL LINES3T3‐L1 mouse fibroblast line
3T3‐L1 (100x) adipocytes (40x)
adipocytes (200x)adipocytes (100x)Differentiated by insulin & IBMX
Deli M
1. Suspension cultures – dilution Blood cells, lymphocytes
2. Adherent cellsEpithelial and endothelial cells
Held together by intercellular junctions (proteins)Junction integrity requires Ca2+, Mg2+
For tissue dissociation and releasing cells:• Chelating agents EDTA• Enzymes trypsin
collagenasedispasepronase
SUBCULTURE OF CELLS(PASSAGE)
WashingIncubating cells in dissociation solutionDispersing (centrifugation)Counting cells: manual, automatedTransfer of cells to new dish
split ratio 1: 2‐6
SUBCULTURE OF CELLS(PASSAGE)
Burker‐chamber for cell counting
FREEZING CELLS FOR STORAGE: DEVICES
Cell freezing Container (Nalgene) ‐ research
isopropanol ‐ slow cooling rate (1ºC / min)
Programmable biofreezer – health industrycooling range: +40ºC/ ‐120 ºC
FREEZING CELLS: CRYOPROTECTORS
Freezing mediacryoprotectors
dimethylsulfoxide (5‐10 % )carboxymethylcelluloseglycerol
STORAGE AND THAWING CELLS
Storage of frozen cell vials
Short term (up to half year)at ‐70 ºC
Long termat ‐198 ºCin cryogenic liquid tanks
Thawingworking quickly warming vials at 37 ºC (water bath)washing the cells – removal of cryoprotectors
MICROBIAL CONTAMINATION OF CULTURES
Sporulating mold colony (60 X)
Bacterial contamination (SEM, 300 X)
Edge of mold colony (300 X, SEM)
Budding yeast (300 X, SEM)
Elimination of yeasts: nystatin, amphotericin‐BElimination of molds: nystatin, amphotericin‐BElimination of bacteria: penicillin, streptomycin, gentamycin, etc.
MICROBIAL CONTAMINATION
Mycoplasma, Hoechst stain
fluorescent microscopy
Mycoplasma pneumoniae (SEM, TEM)
Elimination of mycoplasma:
Erythromycin
Tylosin (Gibco, Sigma)
BM‐cyclin (Roche)
ANTIBIOTICS AND ANTIMYCOTICS FOR CELL CULTURE
antibiotics G+ bacteria G‐ bacteria yeasts molds mycoplasma concentrationampicillin # # 100 μg/mlerythromycin # # 100 μg/mlgentamycin # # # 50 μg/mlkanamycin # # # 100 μg/mllincomycin # 100 μg/mlneomycin # # 50 μg/mlparomomycin # 100 μg/mlpenicillin+streptom. # # 100 U/ml + 100 μg/ml
spectinomycin # # 20 μg/mlspectromycin # # 100 μg/mltetracycline # # 10 μg/ml
tylosin # # 1 μg/ml
antimycoticsamphotericin B # # 2.5 μg/mlnystatin # # 50 μg/ml
M ButlerAnimal cell culture & technologyGarland Science/BIOS Scientific Publishers, 2004
JM DavisBasic cell cultureOxford University Press, 2002
FURTHER READING
CO‐CULTURE MODELS TO STUDY CELL‐CELL INTERACTIONS
• Blood‐brain barrier: an example of cell‐cell interactions• Co‐culture models• Cell‐cell interaction • Conclusion
Brain capillarieslength: 650 km; surface: 10‐20 m2; 0.1% of brain volume
Dynamic interface between the blood and the central nervous systemRoles creates homeostasis for neuronal functions
provides brain with nutrientscommunication between the periphery and the CNSdefense system against toxic insults
BLOOD‐BRAIN BARRIER
TJ
Cross‐talk
Astroglia, pericyte, neuron
Low permeabilityTransporters Receptors
Endothelial cellMetabolic barrier
BBB PROPERTIES ARE ORGAN‐SPECIFIC:CELL‐CELL INTERACTION
FIRST IN VITRO BBB MODELS
Isolation of brain capillaries
Joó and Karnushina, 1973
Living, metabolically active cellsReceptors, transporters, signalling pathwaysGene arrays, proteomic studies
Cultured endothelial cells from brain microvesselsPanula, Joó and Rechardt, 1978
Primary cultures from rat, mouse, bovine, porcine, human, dog, cat, monkey brains
Problem: purity of cultures
INCREASING THE PURITY OF BRAIN ENDOTHELIAL CULTURES
Puromycin treatmentAntibiotics, inhibitor of protein synthesis, P‐glycoprotein substrateBrain endothelium: high P‐glycoprotein level, survives treatmentPericyte: low P‐glycoprotein efflux pump level, cells dye
Perrière et al, J Neurochem., 93: 279‐289, 2005Perrière et al, Brain Res., 1150: 1‐13, 2007
P
E
E
Problem: dedifferentiation, cells lose their properties in cultureCo‐culture of brain endothelial cells with
Astrogliastrengthen barrier functionincrease the expression and activity of enzymes and transporterspart of most co‐culture models
Pericytemesodermal originregulation: blood flow in brain,
endothelial cell division & differentiationvasoactive mediatorsbasal membrane proteinsneuronal stem cell functionBlood‐brain barrier function?
SEM Shepro and Morel, FASEB J, 1993
INDUCTION OF BBB PROPERTIES: CO‐CULTURE
isolation of microvessel fragments
microvascular endothelial cellspuromycin treatment (Perriere et al., 2005)
Rat brain
astrocytes
Rat brain
PREPARATION OF PRIMARY CULTURES
Percoll gradient
MV fractionpericytes
mechanical & enzymic dissociation
MODELS ON CELL CULTURE INSERTS
adherent cells
Resistance Permeability
Cell types:brain endothelial cells, astroglia, pericytes
Double and triple co‐culture
IN VITRO CO‐CULTURE BBB MODELS
Nakagawa et al, Cell Mol Neurobiol., 27: 687‐694, 2007Niwa M, Nakagawa S, Deli MA, WO07072953, 2007Nakagawa et al, Neurochem Int., 54: 253‐263, 2009
brain endothelial cells
astrocytes
pericytes
BBB MODEL – BARRIER FUNCTION
Nakagawa et al, Neurochem Int., 54: 253‐263, 2009
E000
100
200
300
400TEER
(Ω×cm
2 )Day 5
EAP
a, b
EP0a, b, c
EPA
a
E0P
a, b, c
EA0
a, b, c, f
E0A
b, c, d, e
Mono‐culture Double co‐culture Triple co‐culture
APPLICATION OF BBB MODELS
AstrocytePericyte
Pharmacology
Cell interactions Drug effects, ADME
Microbial pathogenesisInvasion of microbes
Disease modelspathomechanism
Endothelial cell
Physiology
Pathology
Cell culture inserts with membranes: co‐culture systems
Co‐culture models: study organ specific cell functions
Blood‐brain barrier = dynamic cooperation: brain endothelial cells, pericytes, astrocytes and neurons
Crosstalk between the cells of the neurovascular unit:blood‐brain barrier phenotype and neuronal functions
CONCLUSIONS
This work is supported by the European Union, co-financed by the European Social Fund, within the framework of " Practice-
oriented, student-friendly modernization of the biomedical education for strengthening the international
competitiveness of the rural Hungarian universities " TÁMOP-4.1.1.C-13/1/KONV-2014-0001 project.