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EP-3545074-B1 - BI-LAYER MULTI-WELL CELL CULTURE PLATFORM

EP3545074B1EP 3545074 B1EP3545074 B1EP 3545074B1EP-3545074-B1

Inventors

  • COPPETA, JONATHAN R.
  • CHAREST, JOSEPH L.
  • VEDULA, ELSE M.
  • BORENSTEIN, JEFFREY T.
  • SPENCER, ABIGAIL JUNE
  • ISENBERG, BRETT C.

Dates

Publication Date
20260506
Application Date
20171121

Claims (14)

  1. An apparatus (100) comprising: a well plate (105) comprising a plurality of structural layers (550, 560, 570), and a membrane (140), wherein the membrane separates two structural layers (560, 570) and the well plate defines an array of tissue modeling environments, and each tissue modeling environment includes: a first fluid reservoir (110a), a second fluid reservoir (110c), a third fluid reservoir (110b) and a fourth fluid reservoir (110d), each fluid reservoir configured to hold a column of fluid; a first microchannel (125a) fluidically coupling the first fluid reservoir to the second fluid reservoir; a second microchannel (125b) fluidically coupling the third fluid reservoir to the fourth fluid reservoir, wherein at least a portion of the first microchannel overlaps at least a portion of the second microchannel across the membrane; and a pump assembly (115) arranged above the well plate and comprising, for each tissue modeling environment: a first output extending down from the pump assembly into the first fluid reservoir for pumping a first fluid into the first fluid reservoir; a first intake extending down from the pump assembly into the second fluid reservoir for pumping the first fluid out of the second fluid reservoir; a second output extending down from the pump assembly into the third fluid reservoir for pumping a second fluid into the third fluid reservoir; and a second intake extending down from the pump assembly into the fourth fluid reservoir for pumping the second fluid out of the fourth fluid reservoir, wherein fluid reservoirs of the array of tissue modeling environments are arranged to correspond to the arrangement of wells of a standard well plate; and wherein the pump assembly is configured to control a first flow rate and a second flow rate for each tissue modeling environment such that the first fluid flows through the first microchannel with the first flow rate and the second fluid flows through the second microchannel with the second flow rate different than the first flow rate.
  2. The apparatus of claim 1, wherein fluid reservoirs of the array of tissue modeling environments are arranged to correspond to the arrangement of wells of a standard 96 well or 384 well well plate.
  3. The apparatus of claim 1, wherein one or more cells are attached to a first side or a second side of the membrane in each tissue modeling environment, wherein the one or more cells attached to the first side of the membrane are renal proximal epithelial cells and the one or more cells attached to the second side of the membrane are endothelial cells.
  4. The apparatus of claim 1, wherein the plurality of structural layers include, a first structural layer, a second structural layer, and a third structural layer, wherein the membrane separates the second structural layer (560) and the third structural layer (570), wherein the first structural layer (550) includes the fluid reservoirs of the tissue modeling environments in the array of tissue modeling environments, the second structural layer defines the first microchannels of the tissue modeling environments in the array of tissue modeling environments, and the third structural layer defines the second microchannels of the tissue modeling environments in the array of tissue modeling environments.
  5. The apparatus of claim 1, wherein the plurality of structural layers includes a first structural layer and a second structural layer, wherein the membrane separates the first structural layer and the second structural layer, and the first structural layer defines the fluid reservoirs and the first microchannels of the tissue modeling environments in the array of tissue modeling environments.
  6. The apparatus of claim 1, wherein the first microchannels or the second microchannels of the tissue modeling environment in the array of tissue modeling environments includes a hydraulic resistor comprising one or more microchannel restrictors.
  7. The apparatus of claim 1, wherein for each tissue modeling environment the first intake is coupled to the first output and the second intake is coupled to the second output.
  8. The apparatus of claim 1, wherein the first or second intake of the pump assembly for at least one tissue modeling environment is coupled to the first or second output of the pump assembly for a different tissue modeling environment.
  9. The apparatus of claim 1, wherein the pump assembly further comprises one of an electromagnetic actuator, a pneumatic actuator, or a hydraulic actuator, and the actuator is configured to induce fluid flow through the pump assembly for a plurality of the tissue modeling environments.
  10. The apparatus of claim 1, wherein the pump assembly includes at least one separate actuator for independently inducing fluid flow through each respective tissue modeling environment.
  11. The apparatus of claim 1, wherein the first microchannel and the second microchannel are defined in an embossed hard plastic, wherein the embossed hard plastic is a cyclic olefin copolymer, COC, fluorinated ethylene propylene, FEP, polymethylpentene, PMP, polyurethane, polystyrene or polysufone.
  12. The apparatus of claim 1, wherein the first microchannel and the second microchannel are formed from a stack of through-cut layers, and wherein the membrane is a track-etched polycarbonate or polyester membrane.
  13. The apparatus of claim 1, further comprising one or more sensor components in each tissue modeling environment, and wherein the sensor components are optical sensors or electrodes (605).
  14. A method for modeling tissue modeling environments using the apparatus of any one of claims 1 to 13, the method comprising: seeding a first cell type into the first microchannel of each tissue modeling environment, seeding a second cell type into the second microchannel of each tissue modeling environment, applying a first feeder flow to the first cell type in the first microchannel of each tissue modeling environment, and applying a second feeder flow to the second cell type in the second microchannel of each tissue modeling environment.

Description

BACKGROUND A well plate, also known as a microtiter plate or microwell plate, is a flat plate with multiple wells used as test tubes or petri dishes for cell culturing and other biomedical experiments and analyses. The industry uses a variety of standard sized well plates. For example, 96 well or 384 well plates are standard tools used for research and diagnostic testing. US2013/059322 discloses microfluidic devices providing for an invasion assay using microfluidic culture systems. US2010/0240086 discloses a biochip assembly comprising a semi-permeable membrane and an assay method using said biochip assembly for carrying out cell based assays. The method involves measuring the migration of cells in a channel under the influence of an analyte wherein the cells are separated from the analyte by a semi-permeable membrane and the analyte and/or the cells are subjected to controlled flow conditions. SUMMARY OF DISCLOSURE According to one aspect, there is provided an apparatus that includes a well plate, which includes a plurality of structural layers and a membrane according to claim 1. According to another aspect, there is provided a method for modeling tissue according to claim 14. BRIEF DESCRIPTION OF THE DRAWINGS The foregoing will be apparent from the following more particular description of example implementations of the invention, as illustrated in the accompanying drawings. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating implementations of the present invention. Figure 1 illustrates an example apparatus for providing an array of tissue modeling environments.Figure 2 illustrates a magnified view of a tissue modeling environment provided by the example apparatus illustrated in Figure 1.Figure 3A illustrates a top down view of the tissue modeling environment illustrated in Figure 2.Figure 3B illustrates a perspective view of the tissue modeling environment illustrated in Figure 2.Figure 3C illustrates a cross sectional view of the tissue modeling environment illustrated in Figure 2.Figure 4A illustrates a top down view of two adjacent tissue modeling environments of the cell culture platform 105 in Figure 1.Figure 4B illustrates the pump assembly interacting with the first and second tissue modeling environments shown in Figure 4A.Figure 5A illustrates an exploded view of the cell culture platform of the example apparatus illustrated in Figure 1.Figure 5B illustrates fluid pathways through the structural layers of the example cell culture platform illustrated in Figure 5A.Figure 6A illustrates an exploded view of an example cell culture platform having an array of tissue modeling environments with integrated sensors.Figure 6B illustrates a top down view of the second structural layer of the example cell culture platform illustrated in Figure 6A.Figure 6C illustrates an exploded view of an example cell culture platform having a single tissue modeling environment with integrated sensors.Figure 6D illustrates a cross sectional view the example cell culture platform shown in Figure 6C.Figure 7A illustrates a cross section of a microchannel structure fabricated by embossing a plastic material.Figure 7B illustrates a cross section of a microchannel structure fabricated using a thru cut technique.Figure 8 illustrates an exploded view of an example cell culture platform fabricated using a thru cut technique, as previously shown in Figure 7B.Figures 9A-9F illustrates top down views of various example implementations of microchannel structures.Figure 10 illustrates views of an example pneumatic manifold actuator and pump assembly.Figure 11 illustrates a flow chart of an example method for populating cells into the cell culture platform of Figure 1.Figure 12 illustrates a flow chart of an example experimental method for simulating hypoxic conditions in healthy tissue using the cell culture platform of Figure 1. For purposes of clarity, not every component may be labeled in every figure. The drawings are not intended to be drawn to scale. Like reference numbers and designations in the various figures indicate like elements. DETAILED DESCRIPTION The various concepts introduced above and discussed in greater detail below may be implemented in any of numerous ways, as the described concepts are not limited to any particular manner of implementation. Examples of specific implementations and applications are provided primarily for illustrative purposes. An industry standard well plate, such as a 96 well plate or a 384 well plate, includes arrays of wells that are used as test tubes or petri dishes. Cells may be deposited into the wells and cultured for further study of microphysiological systems. In microphysiological systems, the rate of change of fluid introduction and withdrawal can have significant effects on media composition and gradients. Providing a constant perfusion of fluid and precisely controlling its flow rate is useful for studying microphysiological systems in a we