Method of osteogenic differentiation in microfluidic tissue culture systems
Inventors
Gazit, Dan • Pelled, Gadi • Gazit, Zulma • Sheyn, Dmitriy • Hinojosa, Christopher David • Wen, Norman • Hamilton, Geraldine
Assignees
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Publication Number
US-12070746-B2
Publication Date
2024-08-27
Expiration Date
Abstract
Microfluidic “organ-on-a-chip” devices have been developed with the aim to replicate human tissues in vitro. However, there is no option to quantitatively monitor biological processes that take place within the chip, over time. Destructive methods in order to analyze, tissue formation, gene expression, protein secretion etc. require the harvest of the “tissue” at a certain time point. Described herein are methods and compositions for non-destructive molecular imaging methods and systems in order to quantitatively monitor specific biological processes, over time, within the chip, without the need to harvest.
Core Innovation
The invention provides a method of osteogenic differentiation using microfluidic “organ-on-a-chip” bone-chip systems. Mesenchymal stem cells (MSCs) are seeded in a microfluidic device in growth medium containing doxycycline (DOX) in the absence of flow to produce attached MSCs, and the attached MSCs are maintained such that proliferation proceeds to produce proliferated MSCs.
During expansion, the growth medium is flowed in the absence of Bone Morphogenetic Protein-2 (BMP2) so that the attached MSCs proliferate to produce proliferated MSCs. The proliferated MSCs are then contacted with an osteogenic medium containing BMP2 to produce differentiated cells that express one or more of osteocalcin, bone sialoprotein (bsp), osteopontin (opn), and collagen type 1.
Constant flow of the osteogenic medium during the contacting step produces a higher level of expression of at least one of osteocalcin, bone sialoprotein (bsp), osteopontin (opn), and collagen type 1 in the differentiated cells compared to in the absence of constant flow. The approach supports non-destructive, longitudinal monitoring without harvesting, including optical imaging of osteogenic differentiation.
Claims Coverage
The document includes one independent claim. It is a single method claim with four inventive features covering DOX-conditioned seeding without flow, BMP2-free proliferation under flow, BMP2-containing osteogenic differentiation, and constant-flow enhancement of osteogenic marker expression.
Microfluidic DOX seeding without flow to produce attached MSCs
Seeding mesenchymal stem cells (MSCs) in a microfluidic device in growth medium containing doxycycline (DOX) in the absence of flow to produce attached MSCs.
Flow expansion in the absence of BMP2 to produce proliferated MSCs
Flowing the growth medium in the absence of Bone Morphogenetic Protein-2 (BMP2) such that the attached MSCs proliferate to produce proliferated MSCs.
BMP2 osteogenic contacting to produce differentiated cells expressing osteogenic markers
Contacting the proliferated MSCs with an osteogenic medium containing Bone Morphogenetic Protein-2 (BMP2) to produce differentiated cells that express one or more of osteocalcin, bone sialoprotein (bsp), osteopontin (opn), and collagen type 1.
Constant flow during osteogenic contacting increases osteogenic marker expression versus no-flow
Providing constant flow of the osteogenic medium during the contacting step to produce a higher level of expression of at least one of osteocalcin, bone sialoprotein (bsp), osteopontin (opn), and collagen type 1 in the differentiated cells compared to in the absence of flowing the osteogenic medium.
The independent claim centers on osteogenic differentiation in a microfluidic device by combining DOX-containing growth medium seeding without flow, BMP2-free expansion under flow, BMP2-containing osteogenic differentiation under constant flow, and higher expression of osteogenic markers versus absence of flow.
Stated Advantages
Enables non-destructive, longitudinal monitoring of osteogenic differentiation without harvesting.
Increases osteogenic marker expression (osteocalcin, bone sialoprotein (bsp), osteopontin (opn), and/or collagen type 1) by providing constant flow of the osteogenic medium compared to absence of flow.
Supports optical imaging of osteogenic differentiation, including luciferase bioluminescence imaging and bisphosphonate fluorescence/near infrared imaging.
Documented Applications
Microfluidic “organ-on-a-chip” bone-chip systems for non-destructive, longitudinal monitoring of osteogenic differentiation of mesenchymal stem cells (MSCs) without harvesting, including optical imaging of osteogenic markers.
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