In vivo, cells in tissues and tumours do not double every 2 – 4
days as they do in classical (2D) cell culture – so why use a technology
developed in the 1950's?
The CelVivo system creates an environment which promotes the growth and
maintenance of large 3D tissue mimetic structures, whether they are
spheroids, organoids, acini and other aggregates.
In this environment, liver spheroids remain in a stable dynamic
equilibrium and exhibit human in vivo physiological performance for at least
24 days (i.e. up to at least 42 days of culture). During this time the
spheroids exhibit a stable transcriptome profile.
Reasons for Using 3D Cell Culture
CELL FEATURE
Advantage over classical 2D cultures For HepG2-C3A - features for other cell lines maybe different
Proliferation
Slows from a doubling time of 1 day to 60 days after 40 days in culture
(see reference 3)
Resembles proliferation in the parental tissue or in tumours
in vivo
Cultures can be maintained for over 300 days
Architecture
Microfilaments (actin) microtubules (tubulin) and intermediate filaments (e.g. keratin) resemble that seen
in vivo(see reference 7)
Ultrastructure
Resembles liver sections, showing plasma membrane differentiation, tight junctions, bile canaliculus-like channels, lipid droplets and glycogen granules
(see reference 2)
Physiological performance
Levels of ATP, urea and cholesterol reach levels seen in vivo
(see reference 3)
Drug metabolism
Toxicity measurements for Amiodorone, APAP, Metformin, Phenformin and Valproic acid are more accurate than those obtained using human primary hepatocytes
(see reference 1)
Proteomics
Numerous changes can be detected throughout the proteome
(see reference 7)
Metabolic reprogramming
> 21 day old spheroids show clear aerobic metabolism (Warburg effect)
(see reference 7)
Epigenetics
Spheroids display histone marks and clipping (not seen using 2D cultures)
(see reference 9)
Integration of cellular response
APAP causes protein oxidation and nitrosylation and this integrates the cellular response to physiological doses of APAP
(see reference 11)
SPHEROIDS
Characteristics For HepG2-C3A - values for other cell lines will be different
Number of cells per spheroid (± 22%)
Day 8 = 23,156
Day 21 = 82,342
Media can be collected via one of the two ports
Spheroid size (Planimetric surface)
Day 8 = 0.26 mm²
Day 21 = 0.58 mm²
Protein concentration
Day 8 = 21.71 µg/ mm²
Day 21 = 43.28 µg/ mm²
ATP concentration
Day 8 = 9.23 µM/ mm²
Day 21 = 8.93 µM/ mm²
Bioreactor soluble protein content
Day 8 = 2,010 µg
Day 21 = 10,120 µg* * the culture will have been split into 4 bioreactors at this stage
BIOREACTOR
Advantages
Easy access to samples and media
The bioreactor can be opened and closed again, providing a petri-dish like access to spheroids and media
Repeated sampling possible
Media can be collected via one of the two ports
Multiple samples
Bioreactor can cultivate up to 300 mature identical 'biopsies' which can either be collected at once for multiple assays or at multiple time points for fewer assays
Dynamic equilibrium
When non-lethal treatment stops, spheroids return to their resting state (dynamic equilibrium)
Optimal growth environment
Rotating bioreactor- clinostat system
Inbuilt humidification chamber with good gas exchange
Inert materials used: non-toxic and low unspecific binding
Use of different cell types
Cell from any species can be used
Primary-, stem- and immortal cells have to be used in clinostat systems
Co-culture of different cell types in the same bioreactor
* The Bioreactor patent is owned by MC2 Biotec (MC2biotec.com).
Celvivo is commercialising it on their behalf.
Left (Video): Changing the growth media in the bioreactors and using the Bioarray matrix drive (BAM).
Right: One of the 16 proprietary drive axel speed controllers.
The drive's control board fits into a standard CO2 incubator (equipped with access port minimum of 24mm in diameter) and has 16 independently control drive axels which can rotate in either direction at speeds from 5 to 50 rpm. The system is temperature inert for the incubator. It can be controlled either from windows or android based devices.
BIOARRAY MATRIX DRIVE
Advantages
Optimal growth environment for 3D culture
Rotating bioreactor- clinostat
Humidified chamber with good gas exchange
Inert materials used: non-toxic and low unspecific binding
Computer controlled multiple drive unit
Does not disturb incubator temperature
Multiple bioreactors
16 independent drives
5-50 rpm in
Easy control
Bioreactor rotation speed can be regulated from
– a tablet (great for working in the cell culture lab)
– a network computer
– or smart phone (using the app provided)
Drives can be regulated independently or in any combination of groups
Easy overview
Large simple display on tablet
Internal log
Quick suspension option
Special start-up program to rapidly disperse spheroids after bioreactor has been stopped (e.g. for medium change or treatment)
