celvivo三维旋转培养系统

型号:
联系人:李胜亮
联系电话:18618101725
品牌:celvivo



3D Cell Culture Technology
Mimicking In vivo Performance

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
Why use 3D Spheroids


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
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
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)

Spheroid Characteristics

Spheroids

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 mm?
  • Day 21 = 0.58 mm?
Protein concentration
  • Day 8 = 21.71 ?g/ mm?
  • Day 21 = 43.28 ?g/ mm?
ATP concentration
  • Day 8 = 9.23 ?M/ mm?
  • Day 21 = 8.93 ?M/ mm?
Bioreactor soluble protein content
  • Day 8 = 2,010 ?g
  • Day 21 = 10,120 ?g*
    * the culture will have been split into 4 bioreactors at this stage

Bioreactor

Bioreactor

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.

BioArray Matrix Drive

 BioArray Matrix 
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)

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
Why use 3D Spheroids


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
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
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)

Spheroid Characteristics

Spheroids

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 mm?
  • Day 21 = 0.58 mm?
Protein concentration
  • Day 8 = 21.71 ?g/ mm?
  • Day 21 = 43.28 ?g/ mm?
ATP concentration
  • Day 8 = 9.23 ?M/ mm?
  • Day 21 = 8.93 ?M/ mm?
Bioreactor soluble protein content
  • Day 8 = 2,010 ?g
  • Day 21 = 10,120 ?g*
    * the culture will have been split into 4 bioreactors at this stage

Bioreactor

Bioreactor

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.

BioArray Matrix Drive

 BioArray Matrix 
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)

* The BAM is at the β-test version stage.



Publications
  1. Metabolic Reprogramming and the Recovery of Physiological Functionality in 3D Cultures in Micro-Bioreactors
    Krzysztof Wrzesinski and Stephen J. Fey
    Bioengineering, 5 (2) 1-25: 2018
    http://www.mdpi.com/2306-5354/5/1/22 
    DOI: 10.3390/bioengineering5010022
    View Publication (PDF)
  2. Recent advances in three-dimensional cell culturing to assess liver function and dysfunction: from a drug biotransformation and toxicity perspective.
    Carlemi Calitz, Josias H. Hamman, Stephen J. Fey, Krzysztof Wrzesinski & Chrisna Gouws
    Toxicology Mechanisms and Methods, 2018
    https://doi.org/10.1080/15376516.2017.1422580 
    DOI: 10.1080/15376516.2017.1422580
    View Publication (PDF)
  3. Acetaminophen-induced S-nitrosylation and S-sulfenylation signalling in 3D cultured hepatocarcinoma cell spheroids.
    K. Wojdyla, K. Wrzesinski, J. Williamson, P. Roepstorff, S.J. Fey, A. Rogowska-Wrzesinska 
    Toxicology Research 5(2) 905-920, 2016 
    http://xlink.rsc.org/?doi=c5tx00469a  
    DOI: 10.1039/C5TX00469A
    View Publication (PDF)
  4. From 2D to 3D - a new dimension for modelling the effect of natural products on human tissue. 
    K. Wrzesinski and S.J. Fey
    Current Pharmaceutical Design 21(38): 5605-5616, 2015. 
    PMID: 26429710 
    DOI: 10.2174/1381612821666151002114227
  5. Top-down and Middle-down Protein Analysis Reveals that Intact and Clipped Human Histones Differ in Post-translational Modification Patterns. 
    A. Tvardovskiy, K. Wrzesinski, S. Sidoli, S.J. Fey, A. Rogowska-Wrzesinska, O.N. Jensen
    Molecular and Cellular Proteomics 14(12):3142-53; 2015. 
    PMID: 26424599 
    DOI: 10.1074/mcp.M115.048975
    Please write to info@celvivo.com for a pdf copy of this publication
  6. The cultural divide: exponential growth in classical 2D and metabolic equilibrium in 3D environments.
    K. Wrzesinski, A. Rogowska-Wrzesinska, R. Kanlaya, K. Borkowski, V. Schw?mmle, J. Daia, K.E. Joensen, K. Wojdyla, V. Botelho Carvalho & S.J. Fey 
    PLOS One 9(9) 1-15; 2014
    PMID: 25222612 
    DOI: 10.1371/journal.pone.0106973
    View Publication (PDF)
  7. The cultural divide: exponential growth in classical 2D and metabolic equilibrium in 3D environments.
    K. Wrzesinski, A. Rogowska-Wrzesinska, K. Borkowski, V. Botelho Carvalho & S.J. Fey 
    Poster at 9th Danish Conference on Biotechnology and Molecular Biology, Vejle; 2014 
    DOI: 10.13140/2.1.2643.2965
  8. Heteromer s – using internal standards to assess the quality of proteomic data.
    A. Rogowska-Wrzesinska, K. Wrzesinski and S.J. Fey
    Proteomics. 14(9):1042-7 2014
    PMID: 24616253 
    DOI: 10.1002/pmic.201300457
    Please write to info@celvivo.com for a pdf copy of this publication.
  9. Microgravity spheroids as a reliable, long term tool for predictive toxicology. 
    S.J. Fey and K. Wrzesinski
    Toxicology Letters, 221S S153 2013. 
    DOI: 10.1016/j.toxlet.2013.05.318
  10. Determination of acute lethal and chronic lethal dose thresholds of Valproic acid using 3D spheroids constructed from the immortal human hepatocyte cell line HepG2/C3A. 
    S.J. Fey and K. Wrzesinski
    In ''Valproic Acid: Pharmacology, Mechanisms of Action and Clinical Implications'' Nova Science Publishers, New York. Ch. V, 141-165; 2013
  11. Human liver spheroids exhibit stable physiological functionality for at least 24 days after recovering from trypsinisation.
    K. Wrzesinski, C.M. Magnone, L. Visby Hansen, M. Ehrhorn Kruse, T. Begauer, M. Bobadilla, M. Gubler, J. Mizrahi, C. M?ller Andreasen, K. Zhang, K. Eyed Joensen, S.M. Andersen and S.J. Fey
    Toxicology Research; 2(3) 163-172; 2013 
    DOI: 10.1039/C3TX20086H 
    NB: Manuscript featured on the front cover of the journal.
    View Publication (PDF)
  12. After trypsinisation, 3D spheroids of C3A hepatocytes need 18 days to re-establish similar levels of key physiological functions to those seen in the liver.
    K. Wrzesinski and S.J. Fey
    Toxicology Research; 2(2) 123-135; 2013 
    DOI: 10.1039/C2TX20060K
    NB: Manuscript featured on the front cover of the journal.
    View Publication (PDF)
  13. Determination of drug toxicity using 3D spheroids constructed from immortalized human hepatocytes. 
    S.J. Fey and K. Wrzesinski
    Toxicological Sciences 127(2) 403-411; 2012. 
    PMID: 22454432 
    DOI: 10.1093/toxsci/kfs122
    View Publication (PDF)