1. Cell and tissue culture

2. Cell culture = propagation of cells outside the organism = in vitro
(Cells in the organism = in vivo)
ADVANTAGES:
cellular environment can be manipulated
well defined cell type
large quantities of cells can be obtained
many cellular functions can be investigated
animal experiments can be replaced
living cells can be tested
human cells can be tested
DISADVANTAGES:
does not mimic the complexity of the human body
spontanous evolution

3. TYPES OF CULTURED CELLS:
primary culture
cell line
finite
continuous
cell strain

4. Primary culture Cells derived directly from tissues
A culture from the time of isolation until its first subculture
First developed in 1907:
axons grow
in culture!
spinal cord explant +
lymphatic fluid
1 day

5. cultured neuron
extending processes

6. Cell line Derived from primary culture via subcultivation (passage)
dissociate
cells
medium
plate cells
tissue explant
cells
dish
Grow and divide while adhering (monolayer) to plastic dishes
Require growth factor for growth
Passaging: after physical or enzymatic dissociation of monolayer cultures the cells are reseeded in lower density

7. Cell line Finite cell line
Finite lifespan
A cell line that survives for a fixed number of population doublings, usually ~40–60 (Hayflick limit), before senescing and ceasing proliferation.

8. Cell line Continuous cell line
Features:
Immortal (indefinite lifespan, over 100 population doublings)
Genetic abnormalities accumulate (transformed cells)
Unlimited growth
Loss of contact inhibition
Loss of adherence dependency (suspension)
injected in mice may form tumor
Transformation can be spontanous or induced by viruses, carcinogens or radiation
May derive:
from finite cell line (in vitro transformation) – e.g. NIH3T3, CHO
from tumors (in vivo transformation) – e.g. HeLa

9. Cell strain
A subpopulation of cell lines that have been purified by physical separation, selection or cloning
Which has specific defined characteristics e.g. marker chromosome, virus resistance, antigen expression

10. normal cells
SEM
transformed cells

11. Cell cultures can be obtained e.g. from cell banks
where these cultures are kept frozen (-196 °C in liquid nitrogen)

12. Density of the culture influences the cell shape

13. Density of the culture influences the cell shape

14. Requirements of cell cultures
Physical parameters
Temperature
O2/CO2.
humidity
Biological parameters
ASEPTIC conditions
Cell density
Co-culture
Metabolites
Feeder cell layer
Chemical parameters
defined medium
serum (growth factors, hormones)
coat on culture surface (collagen, gelatine) pH
osmolarity

15. Laminar box (laminar flow cabinet)
is a carefully enclosed bench designed to prevent contamination of biological samples or any particle sensitive materials.

16. CO2 Incubator
The incubator maintains optimal temperature, humidity and other conditions
such as the carbon dioxide (CO2) and oxygen content of the atmosphere inside.

17. Plasticwares for cell culture

18. Inverted Microscope Light source Objectives
in routine laboratories for live cell inspection

19. R1 ES cells on fibroblast feeder layer
D3 ES (embryonic stem) cells
on fibroblast feeder layer

20. myeloma cells HeLa 53 MONOLAYER CELL SUSPENSION CELL CULTURE CULTURE
Adherent cells, they need surface for proliferation. These are normal cells except hematopoetic cells. Tumor cells may grow in monolayer, too.
SUSPENSION CELL
CULTURE
Non-adherent, floating, generally hemopatopoetic or transformed, tumor cells, they do not need special surface for proliferation.

21. Cell count in function of time
Mozog! sejtmorfológia változása a sejtdenzitás függvényében
Confluent culture

22. Cell count in function of time
3. Stationary phase /plateau
without changing medium
2. log phase
Cell count
subculturing
1. lag phase
time

23. ENVIRONMENTAL INTERACTIONS
Infections (viruses, bacteria,
parasites)
Toxicology
Immunology
Carcinogenesis
Biotransformation of xenobiotics
GENETICS
Transformation
Cell fusion
Cell cycle
BIOCHEMISTRY
DNA transcription
RNA metabolism
Protein synthesis
Intermediate metabolism
BIOTECHNOLOGY/
TISSUE ENGINEERING
Cytokines/growth
factors, hormones,
antibody production
Arteficial tissues
CELL BIOLOGY
Cell-cell and cell-matrix interactions
Gene expression
Cell proliferation
Differentiation
Cell migration, invasion

24. (human cervical cancer)(MGG)
Slide 9. HeLa monolayer
(human cervical cancer)(MGG)
cytoplasm
nucleus
No contact
inhibition
Cell morphology changes
with cell density!!
100% aneuploid.

25. Use and perspectives of cell culture

26. Pluripotent
Pluripotent
Unipotent

28. iPS (induced pluripotent stem) cells

29. Artificial human tissues and organs
Blood vessels - aorta
Liver
Bone
Cartilage
Skin
Retina
Requirements of tissue engineering

30. Human epidermal epithelial cell cultures with
different density and different duration in culture.
Cultured epidermal autograft (e.g. Epicel R)

31. Therapeutic use of human epidermal epithelial cell culture.This photo
was taken 5 years after
the transplantation.

32. Generation of artificial teethor
a scaffold printed in 3D
scaffold may comprise compounds that are chemotactic, osteogenic, dentinogenic, amelogenic, or cementogenic.
Nature Biotechnology 21, (2003)

33. Diagnosis : Amniocentesis – genetic study of the embryo

34. Ex vivo gene therapy
In vivo gene therapy

35. Ex vivo gene therapy in dentristry

36. Facultative slides

37. The immortal life of Henrietta Lacks
1951 Tissue were taken without her knowledge
1952 HeLa cells were used to develop polio vaccine
1955 Isolation of a single cell for cloning
1960 HeLa went to space before any astronaut
1984 HeLa was used to prove that HPV infection
causes cancer
1986 Mechanism of HIV infection were studied
1989 Telomerase were described in HeLa
1993 Tuberculosis was studied
2013 Whole genome data of HeLa were published
born 
Loretta Pleasant in 1920
George Otto Gey

38. History of human embryonic stem cell research
1998 – James Thomson isolated cells from the inner cell mass of the early embryo and developed the first human embryonic stem cell lines
Derivation of the H9 cell line. (A) Inner cell mass–derived cells attached to mouse embryonic fibroblast feeder layer after 8 days of culture, 24 hours before first dissociation. Scale bar, 100 μm. (B) H9 colony. Scale bar, 100 μm. (C) H9 cells. Scale bar, 50 μm. (D) Differentiated H9 cells, cultured for 5 days in the absence of mouse embryonic fibroblasts, but in the presence of human LIF (20 ng/ml; Sigma). Scale bar, 100 μm.
Science  06 Nov 1998:

39. Organ on a chip technique
Microfluidic chamber lined by living human cells
Mimics the interconnectedness of organs within humans
Fields of application:
- disease modelling;
- drug development;
- personalized medicine
Biologically inspired design of a human breathing lung-on-a-chip microdevice. (A) The microfabricated lung mimic device uses compartmentalized PDMS microchannels to form an alveolar-capillary barrier on a thin, porous, flexible PDMS membrane coated with ECM. The device recreates physiological breathing movements by applying vacuum to the side chambers and causing mechanical stretching of the PDMS membrane forming the alveolar-capillary barrier. (B) During inhalation in the living lung, contraction of the diaphragm causes a reduction in intrapleural pressure (Pip), leading to distension of the alveoli and physical stretching of the alveolar-capillary interface. (C) Three PDMS layers are aligned and irreversibly bonded to form two sets of three parallel microchannels separated by a 10-μm-thick PDMS membrane containing an array of through-holes with an effective diameter of 10 μm. Scale bar, 200 μm. (D) After permanent bonding, PDMS etchant is flowed through the side channels. Selective etching of the membrane layers in these channels produces two large side chambers to which vacuum is applied to cause mechanical stretching. Scale bar, 200 μm. (E) Images of an actual lung-on-a-chip microfluidic device viewed from above.

40. A tissue-friendly scaffold covered with self-cells is
not immunogenic.

41. The hydrogel embedded cells are printed in 3D
The hydrogel embedded cells are printed in 3D

42. First artifical trachea