Dynamic interplay of flow and collagen stabilizes primary hepatocytes culture in a microfluidic platform.
The creation of stable flow cultures of hepatocytes is highly desirable for the development of platforms for drug toxicity screening, bio-artificial liver support devices, and models for investigating liver physiology and pathophysiology. Given that hepatocytes cultured using the collagen overlay or in 'sandwich' configuration maintain a wide range of differentiated functions, we describe a simple method for adapting this culture configuration within a microfluidic device. The device design consists of a porous membrane sandwiched between two layers of PDMS resulting in a two-chambered device. In the bottom chamber, hepatocytes are cultured in the collagen sandwich configuration, while the top chamber is accessible for flow. We demonstrate that hepatocytes cultured under flow exhibit higher albumin and urea secretions and induce cytochrome P450 1A1 activity in comparison to static cultures. Furthermore, over two weeks, hepatocytes cultured under flow show a well-connected cellular network with bile canaliculi formation, whereas static cultures show formation of gaps in the cellular network that progressively increase over time. Although enhanced functional response of hepatocytes cultured under flow has been observed in multiple prior studies, the exact mechanism for this flow induced effect remains unknown. In our work, we identified that hepatocytes secrete a higher level of collagen in the flow cultures; inhibiting collagen secretion within the flow cultures reduced albumin secretion and restored the appearance of gaps in the cellular network similar to the static cultures. These results demonstrate the importance of the increased collagen secretion by hepatocytes cultured under flow as a mechanism to maintain a well-connected cellular network and a differentiated function.
Center for Engineering in Medicine (CEM), Massachusetts General Hospital, Harvard Medical School, Shriners Hospital for Children, 51 Blossom Street, Boston, MA 02114, USA. rjindal@mgh.harvard.edu ireis@sbi.org.
Manjunath
M
Hegde
Rohit
R
Jindal
Abhinav
A
Bhushan
Shyam Sundhar
SS
Bale
William J
WJ
McCarty
Inna
I
Golberg
O Berk
OB
Usta
Martin L
ML
Yarmush
D000818:Animals; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods; D002478:Cells, Cultured; D003094:Collagen / Q000737:chemistry; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D004129:Dimethylpolysiloxanes / Q000737:chemistry; D005260:Female; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods; D009757:Nylons / Q000737:chemistry; D051381:Rats; D011917:Rats, Inbred Lew; D012709:Serum Albumin / Q000378:metabolism; D014508:Urea / Q000378:metabolism
D004129:Dimethylpolysiloxanes; D009757:Nylons; D012709:Serum Albumin; C085707:poly(dimethylsiloxane)-polyamide copolymer; D014508:Urea; D003094:Collagen; D019363:Cytochrome P-450 CYP1A1