A microfabricated array bioreactor for perfused 3D liver culture.
We describe the design, fabrication, and performance of a bioreactor that enables both morphogenesis of 3D tissue structures under continuous perfusion and repeated in situ observation by light microscopy. Three-dimensional scaffolds were created by deep reactive ion etching of silicon wafers to create an array of channels (through-holes) with cell-adhesive walls. Scaffolds were combined with a cell-retaining filter and support in a reactor housing designed to deliver a continuous perfusate across the top of the array and through the 3D tissue mass in each channel. Reactor dimensions were constructed so that perfusate flow rates meet estimated values of cellular oxygen demands while providing fluid shear stress at or below a physiological range (<2 dyne cm(2)), as determined by comparison of numerical models of reactor fluid flow patterns to literature values of physiological shear stresses. We studied the behavior of primary rat hepatocytes seeded into the reactors and cultured for up to 2 weeks, and found that cells seeded into the channels rearranged extensively to form tissue like structures and remained viable throughout the culture period. We further observed that preaggregation of the cells into spheroidal structures prior to seeding improved the morphogenesis of tissue structure and maintenance of viability. We also demonstrate repeated in situ imaging of tissue structure and function using two-photon microscopy.
Powers
Mark J
MJ
Division of Bioengineering & Environmental Health, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Domansky
Karel
K
Kaazempur-Mofrad
Mohammad R
MR
Kalezi
Artemis
A
Capitano
Adam
A
Upadhyaya
Arpita
A
Kurzawski
Petra
P
Wack
Kathryn E
KE
Stolz
Donna Beer
DB
Kamm
Roger
R
Griffith
Linda G
LG
D000818:Animals; D019149:Bioreactors; D018929:Cell Culture Techniques; D049452:Green Fluorescent Proteins; D022781:Hepatocytes / Q000166:cytology / Q000502:physiology*; D021621:Imaging, Three-Dimensional; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008297:Male; D008853:Microscopy; D009024:Morphogenesis / Q000502:physiology; D009924:Organ Culture Techniques / Q000379:methods / Q000639:trends; D010100:Oxygen / Q000502:physiology; D010477:Perfusion; D051381:Rats; D013314:Stress, Mechanical; D023822:Tissue Engineering / Q000379:methods* / Q000639:trends
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D010100:Oxygen