Interwoven four-compartment capillary membrane technology for three-dimensional perfusion with decentralized mass exchange to scale up embryonic stem cell culture.
We describe hollow fiber-based three-dimensional (3D) dynamic perfusion bioreactor technology for embryonic stem cells (ESC) which is scalable for laboratory and potentially clinical translation applications. We added 2 more compartments to the typical 2-compartment devices, namely an additional media capillary compartment for countercurrent 'arteriovenous' flow and an oxygenation capillary compartment. Each capillary membrane compartment can be perfused independently. Interweaving the 3 capillary systems to form repetitive units allows bioreactor scalability by multiplying the capillary units and provides decentralized media perfusion while enhancing mass exchange and reducing gradient distances from decimeters to more physiologic lengths of <1 mm. The exterior of the resulting membrane network, the cell compartment, is used as a physically active scaffold for cell aggregation; adjusting intercapillary distances enables control of the size of cell aggregates. To demonstrate the technology, mouse ESC (mESC) were cultured in 8- or 800-ml cell compartment bioreactors. We were able to confirm the hypothesis that this bioreactor enables mESC expansion qualitatively comparable to that obtained with Petri dishes, but on a larger scale. To test this, we compared the growth of 129/SVEV mESC in static two-dimensional Petri dishes with that in 3D perfusion bioreactors. We then tested the feasibility of scaling up the culture. In an 800-ml prototype, we cultured approximately 5 x 10(9) cells, replacing up to 800 conventional 100-mm Petri dishes. Teratoma formation studies in mice confirmed protein expression and gene expression results with regard to maintaining 'stemness' markers during cell expansion.
Gerlach
Jörg C
JC
Department of Surgery, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15203, USA. joerg.gerlach @ cellnet.org
Lübberstedt
Marc
M
Edsbagge
Josefina
J
Ring
Alexander
A
Hout
Mariah
M
Baun
Matt
M
Rossberg
Ingrid
I
Knöspel
Fanny
F
Peters
Grant
G
Eckert
Klaus
K
Wulf-Goldenberg
Annika
A
Björquist
Petter
P
Stachelscheid
Harald
H
Urbaniak
Thomas
T
Schatten
Gerald
G
Miki
Toshio
T
Schmelzer
Eva
E
Zeilinger
Katrin
K
D000818:Animals; D019149:Bioreactors; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D048708:Cell Growth Processes / Q000502:physiology; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology*; D006801:Humans; D051379:Mice; D010477:Perfusion