Estabilización de la célula con los procesos fisiológicos

Regulación de la estabilidad interna

Regulación osmótica
Excreción
Regulación de la temperatura
Regulacion de pH
 Tissue Culture

Ross Granville Harrison 1907

Chose the frog
cold-blooded animal
Incubation was not required

Marie Joseph Auguste (Alexis) Carrel 1912

Developed a methodology for maintaining cultures free from
contamination, especially by bacteria.

Method for studying the behavior of animal cell

 Classification of tissue culture

Organ culture

Three-dimensional culture
Retaining some or all histological features

Tissue culture

Undisaggregated fragments of tissue

Cell culture

Derived from dispersed cells taken from the original tissue

Areas of interest in tissue culture

Intracellular activity
DNA transcription
Protein synthesis

Intracelluar flux
RNA, hormones, metabolites

Ecology
Infection
Drug action
Population kinetics
Membrane flux

Cell-Cell interaction
Contact inhibition
Limitation of grow
Embryonic induction
Advantages of tissue culture
Control of physiochemical environmental
(pH, temperature, osmotic pressure, O2, CO2)

Constitution of homogeneous cell

Disadvantages
Easy to grow many contaminants
Complex environmental
Production of relatively little tissue
Instability of chromosomal constitution
 Adult animal Embryo Egg

Dissection

Enzyme digestion
Finely chopped

Cell culture
Primary explants Organ culture
Initiation of the culture

Capacity to migrate from the explants

Survive to disaggregation technique
Adhere to the monolayer
Survive in suspension
 Primary culture
Evolution of a cell line Continuous cell line
Cell Line

20

18

16
Log 10 cell no. Transformation

14

1st Subculture
Explantation
Senescence
12
and death

10
Subculture interval
8
Serial passage
5

Weeks in culture
Primary tissue culture

• A culture derived directly from a tissue

• Best resembling natural tissue
• Limited growth potential
• May give rise to a cell strain (Cell line)
or be immortalized
• Cell line is a lineage of cells
originating from one primary
culture
 Steps in primary tissue culture

Origin of tissue

Primary explant

Disggreggation of tissue

Iniciation of culture

INCUBATION
 Disaggregation of cells

• Cells can migrate out from an explant

• Mechanical dissociation (mincing)
• Enzymatic dissociation
Exception – hematopoietic cells
Explant culture

• piece of tissue into the tissue culture dish

•Cells migrate out from the tissue
• First type of cell culture developed
• Or in case of small amount of tissue (such as needle
biopsies)
• Not very effective for cells with poor adhesion
(migration)
• Fibrinogen and thrombin used to stimulate adhesion
 Enzymatic disaggregation

• Cell to cell adhesion is mediated by a variety of cell adhesion molecules

• The connections between cells and extracellular matrix
have to be broken
• To break calcium dependent adhesion (cadherins and
selectins) require EDTA or EGTA (both calcium chelators)
• Extracellular matrix proteins such as fibronectin and
laminin are protease sensitive
• Proteoglycans can be partially degraded by hyaluronidase
or heparinase
 Physical connections between cells

• Cells in multicellular organisms are in contact with each other or extracellular matrix
• Cell connections involve multiple ligands and cell adhesion receptors
• The interaction between cell adhesion receptors and their ligands are relatively weak
Enzymes used in enzymatic disaggregation

– Trypsin
– Collagenase
– Elastase
– Hyaluronidase
Cultivos Primarios
 Fibroblastos
humanos
400 X

Aislados mediante digestión

enzimática de una biopsia de
piel de un varón caucasiano.
Mantenidos en cultivo hasta
pase 12 a 15.

200 X
 Queratinocitos humanos
queratinocitos humanos. Aislados
mediante digestión enzimática. Se
cultivan sobre una capa basal de
alimentación ('feeder layer') de
fibroblastos 3t3-SA tratados con
mitomicina

200 X
 Hepatocitos de Rata

hepatocitos de rata.Aislados mediante

colagenasa. Mantenidos hasta 2
semanas en presencia de factores de
diferenciación. Los hepatocitos de rata
adulta no proliferan, a diferencia de
los aislados de hígado neonatal.

400 X
 Hepatocitos humanos

hepatocitos humanos. con colagenasa a

partir de biopsias

400 X
 Epitelio Pigmentario de Retina Humano

células del epitelio pigmentario de

la retina humana (HRPE). Aislados
mediante digestión con colagenasa
intraocular

400 X
 Células endoteliales de Cordón Umbilical
Humano

Obtenidas por digestión con colagenasa de vasos de cordón umbilical humano