3 Biotec

Ingeniería Biomédica Biotecnología en salud
CELL CULTURE
„Cell culture is a process of isolating cells from the organism
and their growth in the controlled artificial conditions
– out of their natural environment – in vitro.“
fungi
plants
animals
yeast (mammals & insects)
+
humans
bacteria
https://bit.ly/37n6vY7
How will you get your cell line from an organism/organ?
https://bit.ly/3dIfrs1
https://bit.ly/2To2nyZ https://bit.ly/35dzpXS
https://bit.ly/2To2nyZ
https://bit.ly/2H9gwO1
Adipose tissue-derived
mesenchymal stem/stromal cells
https://bit.ly/31mIwEI
Ingeniería Biomédica LT1
Biotecnología en salud
Mechanical disaggregation
1. Pressing the piece of tissue through
straws with a gradual decrease of
the holes
https://bit.ly/2HyUSCg
2. Forcing the tissue fragments https://bit.ly/31RdoNV

through a syringe and needle
3. Dissociators
- used for soft tissues – brain, soft

tumors, spleen
https://bit.ly/31z3oZn https://bit.ly/2HtD0ZE
Snímka 36
LT1 Lenka Toro, 23/10/2020

Mechanical disaggregation
author unknown
https://bit.ly/3mjUFSz
https://bit.ly/2HtD0ZE
Risk – damaging cells, loss of small sample, decrease of viability of cells
Advantage – less expensive, quick and simple method

Enzymatic disaggregation
Trypsin – dissolves proteins in ECM (protease); (term: trypsinization)
1. warm (37 °C) – shorter time of incubation (30 min – hours),
mixing, centrifugation required
lower yield, lower viability of cells
https://bit.ly/3dSyErc
2. cold (4 °C ) – longer incubation (6-24 h), no mixing, no
centrifugation,
higher yield, higher viability of cells, can be done overnight
Collagenase – the ECM containing collagen – connective tissue and

muscle
Hyaluronidase – dissolves proteoglycans in ECM
DNase – dissolves DNA aggregates from damaged cells
Combinations – trypsin:accutase or trypsin:collagenase
single cell suspension
Advantage – high viability and recovery of cells
https://bit.ly/3onG3no
Explant technique
- tissue is minced to smaller pieces

- small volume of media
- tissue cannot float!! Lenka Toro https://bit.ly/3kpewzt
- after 3-5 days of incubation the media can be added/changed
- incubating until the outgrowth of cells is observed
- the explants can be transferred to a new culture plastic or
removed and cells subcultured
https://bit.ly/3dRuUWR
https://bit.ly/3j9zzEE
1. Primary cell culture

Process of selection due to
migration and adherence, later due
to their ability to proliferate
Types of primary culture

- mouse/chicken embryos https://bit.ly/34nG2YA
- chick embryo organ rudiments
(brain, heart, lungs, liver, kidney,
skin...)
- transplantable animal tumors
- human biopsy material
(macrophages do not divide in vitro

and can be used as primary culture) https://bit.ly/3joR6Zq
https://bit.ly/34lvid2
First step of culturing cells – freshly isolated cells from the tissue and placed into suitable
controlled conditions (medium, temperature, pH...) – until they consume all the nutrients
necessary for their growth
Separation techniques – size of the cells, surface markers – antibodies – immunosorting, specific growth medium
or
heterogenous population of cells
https://bit.ly/2Td1tVS
Confluence: proportion of the surface (dish, flask) covered by cells
adherent
vs.
suspension
cells and
their
confluence
https://bit.ly/31Cv7rY
PRIMARY CELL CULTURE
+ Advantages
- usually keeps many of the differentiated characteristics of the cell in vivo (parental morphology)
- the best experimental model for in vivo situations
- Disadvantages
- Difficult to obtain – slow proliferation
- Often contaminated by bacteria https://bit.ly/2Td1tVS
- Short lifespan in vitro – limited period of time - senescence

- Not well characterized
- Preparation and maintainance is long and expensive
- Heterogeneous - it can overgrown by other cells (Schwann cells/fibroblasts)
2. Cell lines
Permanently established cell culture that will proliferate indefinitely under proper
conditions (physical & chemical, space)
www.atcc.org
2. Cell lines
Permanently established cell culture that will proliferate indefinitely under proper conditions (physical & chemical, space)
Subculture (Passage)
– removal of the media and transfer of cells into a fresh medium to prevent the reduction of cell proliferation
https://bit.ly/3dSEU2a
https://bit.ly/2Td1tVS https://bit.ly/34nPfQI
CELL LINES
a, finite
- capable of only limited number of population doublings – senescence (≈30 cycles of division)
- Hayflick´s limit - shortening of telomeres and factors regulating the growth cycle (p53, pRb)
- euploid/diploid (human – 2x 23 chromozomes)
- anchorage dependent (adherence to other cells or substrate (matrix) or plastic)
b, continous
Fibroblasts migration
https://www.youtube.com/watch?v=609SS3NM0nI&ab_channel=Radboudumc
Anchorage dependence – Adherent cell lines
- all normal (non-transformed) tissue-derived cells are anchorage dependent except the cells
from hematopoietic origin
- for normal proliferation/differentiation cells need

(1) cell-cell (tight/gap junctions)
(2) cell-ECM (integrins) https://bit.ly/3medwOV
(3) cell-plastic surface/support
human bronchial epithelial cells

- absence of a support leads to arrest in cell growth
https://bit.ly/31BPuWq
anoikis – (Greek; homelessness)

- a form of programmed cell death induced by
anchorage-dependent cells detaching from ECM
- prevents re-adhesion of detached cells to incorrect location
https://bit.ly/3kjA5RQ
Anchorage independence – suspension cells
- cells derived from hematopoietic origin or transformed/tumor cells

- grow in suspension: non-adhesive
- cells maintained in vitro can require agitation (mixing) – for gas exchange in media
https://bit.ly/3og1Mxv https://bit.ly/37AdwVm
Cell lines
a, finite
- capable of only limited number of population doublings – senescence (≈30 cycles of division)
- Hayflick´s limit - shortening of telomeres and factors regulating the growth cycle (p53, pRb)
- euploid/diploid (human – 2x 23 chromozomes)
- anchorage dependent (adherence to other cells or substrate (matrix) or plastic)
- growth rate – slower
- cloning efficiency – low
- serum requirement - high
- human fibroblasts (50 cycles of division, euploid), glial cells
b, continous
Cell lines
b, continous
- capable of unlimited number of population doublings (immortal cell
culture)
- transformed to immortal cell line (chemically, viral oncogenes, tumor
cells, spontanously)!!!
- overcame Hayflick´s limit – enzyme telomerase prolongs telomeres -
immortality
- heteroploid/aneuploid
- growth: in monolayer (anchorage dependent) or in suspension (a. i.)
- growth rate – faster
- cloning efficiency – high
- serum requirement - low
- melanoma (A375), breast cancer, ovarian carcinoma...
HeLa cells – immortalized cell line – abnormal division (no. of chromosomes: 82!)
https://bit.ly/3omXgwS
https://bit.ly/3jqdgL3
Finite cell line
Continuous
cell line
https://bit.ly/35mx90y
CELL LINES
- Disadvantages
- they differ from the primary cultures –very little original in vivo features
- continuous cell lines – tumorigenic (genetic changes, overcoming cell cycle arrest..)
+ Advantages
- Easy to obtain (ATCC, ECACC - European Collection of Authenticated Cell Cultures)
- Fast proliferation
- Mycoplasma/Bacteria free from the cell bank
- Continuous c.l. - unlimited number of division (HeLa – 1951-2018)
- Very well characterized
- Preparation and maintainance is shorter and less expensive
- Homogeneous, one cell population (cross-contamination!)
Why do we need cell cultures?
- reduce animal use!

- to study: normal physiology and biochemistry of cells
- reducing the effect of the surrounding tissues
- reducing the variations caused by stress in animals in experiments
- drug screening and development, testing the toxic compounds
- to study mutagenesis and carcinogenesis (anticancer research, genetic disorders, gene therapy)
- genetic manipulation – understanding the behavior in the organism
- tissue engineering – regenerative medicine
- in vitro fertilization
Why do we need cell cultures?

CELL CULTURE
- commercial use – production of material – vaccines, antibodies, hormones, proteins...
https://go.nature.com/3dREs49

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Ingeniería Biomédica Biotecnología en salud

How will you get your cell line from an organism/organ?

2. Forcing the tissue fragments https://bit.ly/31RdoNV

- used for soft tissues – brain, soft

LT1 Lenka Toro, 23/10/2020

Risk – damaging cells, loss of small sample, decrease of viability of cells

Advantage – less expensive, quick and simple method

Collagenase – the ECM containing collagen – connective tissue and

- tissue is minced to smaller pieces

1. Primary cell culture

Types of primary culture

(macrophages do not divide in vitro

1. Primary cell culture

1. Primary cell culture

Confluence: proportion of the surface (dish, flask) covered by cells

- Short lifespan in vitro – limited period of time - senescence

Anchorage dependence – Adherent cell lines

- for normal proliferation/differentiation cells need

human bronchial epithelial cells

- absence of a support leads to arrest in cell growth

anoikis – (Greek; homelessness)

Anchorage independence – suspension cells

- cells derived from hematopoietic origin or transformed/tumor cells

Why do we need cell cultures?

- reduce animal use!

Why do we need cell cultures?

- commercial use – production of material – vaccines, antibodies, hormones, proteins...

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