elŐadÁs kivonat classroom lecture handout … · erythromycin tylosin (gibco, sigma) bm ‐cyclin...

TÁMOP-4.1.1.C-13/1/KONV-2014-0001 projekt

Biological Research Centre Address: H-6726 Szeged, Temesvári krt. 62. Mail: H-6701 Szeged, POB 521. www.brc.hu

„Az élettudományi-klinikai felsőoktatás gyakorlatorientált és hallgatóbarát korszerűsítése a vidéki képzőhelyek nemzetközi versenyképességének erősítésére”

program keretében finanszírozott

ELŐADÁS KIVONAT

CLASSROOM LECTURE HANDOUT

financed by the program

„Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities”

Dátum / Date:

FEBRUARY 27, 2019

Helyszín / Place:

MTA SZBK BIOFIZIKAI INTÉZET, TANÁCSTEREM / LECTURE ROOM, INST. OF BIOPHYSICS, BIOLOGICAL RESEARCH CENTRE

SZEGED, TEMESVÁRI KRT. 62.

Az előadás címe / Title of the presentation:

MAMMALIAN CELL CULTURE AND CO-CULTURE MODELS

Előadó / Speaker:

MÁRIA A. DELI

M. Deli February 27, 2019

1

CO‐CULTURE MODELS TO STUDYMAMMALIAN CELL INTERACTIONS

Maria A. Deli

Institute 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


2

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


3

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 centrifugeVacuum pumpBottles: used medium

humidity trap


4

Cabinet for sterilized glassware and culture dishes

Pipet aids

EQUIPMENTS FOR CELL CULTURE

Glassware

pipets, 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 °C

metal, glass (eg. surgical instruments, glass pipettes)

2. Autoclaving 15‐30 min, 1 atm, 121 °C

liquids, bottles with cap, plasticware, papers, textiles

3. Gas sterilization ethylene oxide (hospitals), formaldehyde

instruments, catheters, big equipment, rooms

4. Irradiation 60 Co γ‐irradiation

commercial TC plasticware

5. Filtration 0.45 μm, 0.2 μm, 0.1 μm, different membranes

assembling / disposable

buffers, protein solutions

Control of sterilization indicator tapes

microbiological culture

STERILIZATION


5

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


6

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‐5

Walter 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


7

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., 1996

SV40 large T antigen

• RBE4 (rat)Durieu‐Trautmann et al., 1993

Adenovirus‐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


8

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 trypsincollagenasedispasepronase

SUBCULTURE OF CELLS(PASSAGE)

Washing

Incubating cells in dissociation solution

Dispersing (centrifugation)

Counting cells: manual, automated

Transfer of cells to new dish

split ratio 1: 2‐6

SUBCULTURE OF CELLS(PASSAGE)

Burker‐chamber for cell counting


9

FREEZING CELLS FOR STORAGE: DEVICES

Cell freezing CoolCell container (BioCision) ‐ research

polyethylene foam insulating housethermo‐conductive solid state core 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


10

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 concentration

ampicillin # # 100 μg/ml

erythromycin # # 100 μg/ml

gentamycin # # # 50 μg/ml

kanamycin # # # 100 μg/ml

lincomycin # 100 μg/ml

neomycin # # 50 μg/ml

paromomycin # 100 μg/ml

penicillin+streptom. # # 100 U/ml + 100 μg/ml

spectinomycin # # 20 μg/ml

spectromycin # # 100 μg/ml

tetracycline # # 10 μg/ml

tylosin # # 1 μg/ml

antimycotics

amphotericin B # # 2.5 μg/ml

nystatin # # 50 μg/ml


11

M Butler

Animal cell culture & technologyGarland Science/BIOS Scientific Publishers, 2004

JM Davis

Basic 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 capillaries

length: 650 km; surface: 10‐20 m2; 0.1% of brain volume

Dynamic interface between the blood and the central nervous system

Roles creates homeostasis for neuronal functions

provides brain with nutrients

communication between the periphery and the CNS

defense system against toxic insults

BLOOD‐BRAIN BARRIER


12

TJ

Cross‐talk

Astroglia, pericyte, neuron

Low permeabilityTransporters Receptors

Endothelial cell

Metabolic 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 cells

Receptors, transporters, signalling pathways

Gene 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 treatment

Antibiotics, inhibitor of protein synthesis, P‐glycoprotein substrate

Brain endothelium: high P‐glycoprotein level, survives treatment

Pericyte: 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


13

Problem: dedifferentiation, cells lose their properties in culture

Co‐culture of brain endothelial cells with

Astrogliastrengthen barrier functionincrease the expression and activity of enzymes and transporterspart of most co‐culture models

Pericyte

mesodermal 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


14

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

400

TEER

(Ω×cm

2)

Day 5

EAP

a, b

EP0

a, 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


15

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

elŐadÁs kivonat classroom lecture handout … · erythromycin tylosin (gibco, sigma) bm ‐cyclin...

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