Pumpless platform for high-throughput dynamic multicellular culture and chemosensitivity evaluation
Inventors
Lee, Woo • CHEN, Zhehuan • Zilberberg, Jenny
Assignees
Hackensack Meridian Health Center For Discovery And Innovation • Stevens Institute of Technology
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Publication Number
US-11421194-B2
Publication Date
2022-08-23
Expiration Date
Abstract
The described invention provides an ex vivo dynamic multiple myeloma cancer niche contained in a pumpless perfusion culture device. The dynamic multiple myeloma cancer niche includes (a) a three-dimensional tissue construct containing a dynamic ex vivo bone marrow niche, which contains a mineralized bone-like tissue containing viable osteoblasts self-organized into cohesive multiple cell layers and an extracellular matrix secreted by the viable adherent osteoblasts; and a microenvironment dynamically perfused by nutrients and dissolved gas molecules; and (b) human myeloma cells seeded from a biospecimen composition comprising mononuclear cells and the multiple myeloma cells. The human myeloma cells are in contact with osteoblasts of the bone marrow niche, and the viability of the human myeloma cells is maintained by the multiple myeloma cancer niche.
Core Innovation
An in vitro multiwell plate-based pumpless perfusion culture device comprises a bottomless multi-well plate with four adjacent sequential wells forming a culture chamber, including an inlet well, a cell seeding port well, a cell chamber well, and an outlet well. The device includes layered micropatterned pressure-sensitive adhesive (PSA) and a polymer layer with vertically aligned holes, together with an intermediate layer that includes first and second polymer membranes.
The layered construction further includes a first micropatterned PSA layer attached to a bottom surface of the culture chamber, a second micropatterned PSA layer attached to a bottom surface of the polymer layer, and a third micropatterned PSA layer that connects the four adjacent sequential wells. A transparent, optical grade glass layer is attached to the bottom surface of the third micropatterned PSA layer to form a bottom surface and seal the device. The inlet and outlet wells act as reservoirs to generate hydrostatic pressure differential, while the first micropatterned PSA layer maintains stable liquid droplets and prevents medium evaporation by covering the inlet, cell seeding port, and outlet wells.
The invention also provides an ex vivo multiple myeloma cancer niche contained in the microfluidic device, where flow of minute amounts of liquids or dissolved gas molecules is controlled by microfluidics. The niche includes an ex vivo bone marrow microenvironment perfused by nutrients and dissolved gas molecules, with viable osteoblasts seeded to form 3D nodular structures comprising 3D bone-like tissue characterized by extracellular matrix secreted by the viable osteoblasts.
A multiple myeloma tumor biospecimen comprising viable human multiple myeloma cells is provided, optionally with autologous plasma, and is brought into contact with osteoblasts to form an ex vivo microenvironment recapitulating spatial and temporal characteristics of a multiple myeloma cancer niche in vivo while maintaining viability of the multiple myeloma cells.
Claims Coverage
The provided material includes four independent claims, corresponding respectively to the pumpless perfusion culture device, an ex vivo multiple myeloma cancer niche in a microfluidic device, a method for preparing the ex vivo multiple myeloma cancer niche, and a method for assessing chemotherapeutic efficacy using the ex vivo multiple myeloma cancer niche. The claims center on a layered micropatterned PSA/polymer pumpless perfusion architecture, an osteoblast-based bone marrow niche, and viable human multiple myeloma cells responsive to perfusion conditions.
Layered pumpless perfusion culture device with rocking platform
An in vitro multiwell plate-based pumpless perfusion culture device comprising a bottomless multi-well plate with four adjacent sequential wells forming a culture chamber, layered micropatterned PSA and polymer layers with vertically aligned holes, a transparent optical grade glass sealing layer, and a rocking platform holding the plate with rocking speed and an adjustable rocking/tilt angle, wherein inlet and outlet wells generate hydrostatic pressure differential and the device maintains stable liquid droplets and prevents medium evaporation.
Ex vivo multiple myeloma cancer niche with microfluidic perfusion and viable osteoblast-derived bone-like tissue
An ex vivo multiple myeloma cancer niche contained in a device in which flow of minute amounts of liquids or dissolved gas molecules is controlled by microfluidics, comprising an ex vivo bone marrow microenvironment perfused by nutrients and dissolved gas molecules with viable osteoblasts forming 3D nodular structures comprising 3D bone-like tissue, and a multiple myeloma tumor biospecimen with viable human multiple myeloma cells, wherein the niche is effective to recapitulate spatial and temporal characteristics of a multiple myeloma cancer niche in vivo and to maintain viability of the myeloma cells ex vivo.
Preparing an ex vivo multiple myeloma cancer niche by perfused osteoblast tissue formation and seeding patient-derived cells
A method for preparing an ex vivo multiple myeloma cancer niche contained in a device, wherein microfluidics controls flow of minute amounts of liquids or dissolved gas molecules, including constructing a perfused bone marrow microenvironment by seeding viable osteoblasts and culturing to form 3D nodular structures comprising 3D bone-like tissue, preparing a multiple myeloma tumor biospecimen composition comprising viable human multiple myeloma cells from a subject and plasma autologous to the subject, and seeding the perfused bone marrow microenvironment with the biospecimen composition to form an ex vivo microenvironment recapitulating spatial and temporal characteristics of a multiple myeloma cancer niche in vivo and maintaining viability of the myeloma cells.
Chemotherapeutic efficacy assessment by comparing viability and apoptosis in the perfused ex vivo niche
A method for assessing chemotherapeutic efficacy of a test chemotherapeutic agent on viable human multiple myeloma cells seeded in an ex vivo microenvironment that recapitulates spatial and temporal characteristics of a multiple myeloma cancer niche and maintains viability, comprising contacting the ex vivo multiple myeloma cancer niche with a test chemotherapeutic agent and comparing at least one of viability and level of apoptosis to an untreated control.
Across the independent claims, the main inventive features are a layered micropatterned PSA/polymer pumpless perfusion architecture sealed with an optical-grade glass layer and used on a rocking platform, and the formation of an osteoblast-derived perfused 3D bone-like niche that supports an ex vivo multiple myeloma cancer niche. The niche is responsive to microfluidic perfusion and is used to maintain viability of viable human multiple myeloma cells and to assess chemotherapeutic efficacy by comparing viability and apoptosis against an untreated control.
Stated Advantages
maintain stable liquid droplets and prevent medium evaporation
control flow of minute amounts of liquids, nutrients and dissolved gas molecules
generate hydrostatic pressure differential between wells
control the flow rate of medium in the culture chamber
hold the medium within the culture chamber during cell seeding and culture
recapitulate spatial and temporal characteristics of a multiple myeloma cancer niche in vivo
maintain viability of the multiple myeloma cells in the microfluidic device ex vivo
assess chemotherapeutic efficacy by comparing viability and level of apoptosis to an untreated control
Documented Applications
Preparation of an ex vivo multiple myeloma cancer niche in an osteoblast-derived 3D bone-like tissue microenvironment perfused by nutrients and dissolved gas molecules using the described pumpless microfluidic device.
Recapitulating in vivo spatial and temporal characteristics of a multiple myeloma cancer niche and maintaining viability of multiple myeloma cells ex vivo.
Assessment of chemotherapeutic efficacy of a test chemotherapeutic agent on viable human multiple myeloma cells maintained in the ex vivo microenvironment by comparing viability and/or apoptosis versus an untreated control.
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