System and method for preparing three-dimensional patterns for culturing cells

Abstract

Systems for preparing a three-dimensional pattern to enable an aligned growth of cultured cells are provided. The system can include a patterning layer holder including an elongated body having a patterning layer engaging portion provided in a distal region thereof and sized for insertion into a cell culture well, and a peripheral wall defining a patterning layer cavity, the patterning layer cavity being configured to house therein at least a proximal portion of a patterning layer. The peripheral wall can further define a longitudinally-extending through-hole configured to receive a patterning layer-creating material in a pre-cast form to subsequently cast the patterning layer within the patterning layer cavity. A plurality of patterning layer holders can be joined via a common top platform. Methods for preparing first and second three-dimensional patterns on first and second pattern-receiving surfaces of first and second cell culture wells are also provided.

Description

SYSTEM AND METHOD FOR PREPARING THREE-DIMENSIONAL PATTERNS FOR CULTURING CELLSTECHNICAL FIELD

[0001] The technical field generally relates to cell culture techniques. More particularly, the technical field relates to techniques for preparing three-dimensional patterns for culturing cells according to an organized network.BACKGROUND

[0002] Models that can predict human or animal responses to various chemical and biological compounds, such as medications, pesticides, and cosmetics, and that are easily scalable to test multiple compounds already on the market and new products launched each year are desirable.

[0003] In vitro models can be used to refine, reduce, and replace animal testing, for instance in the pharmaceutical, chemical, and cosmetic industries. In vitro models can offer opportunities for performing various experiments on biological materials. These experiments can be performed for instance in relation to toxicity testing, efficacy testing, drug screening, drug repurposing, disease modelling, etc.

[0004] However, the preparation of in vitro models for certain types of cells can prove challenging. For instance, the cell culture of cardiomyocytes in standard cell culture dishes or standard cell culture plates may result in poor sarcomere organization since cultured cardiomyocytes can adopt a random configuration. Such random configuration of the cultured cardiomyocytes can result in models that may not accurately mimic the cardiac tissue structure and the functional feedback from the cardiac tissue, and thus do not mimic the in vivo behaviour of cardiomyocytes in heart tissue. In turn, models that do not mimic the in vivo behaviour of cardiomyocytes can prevent, for instance, assessing electrochemical propagation and render the model unsuitable for performing tests or assays in relation to the electrical, chemical, or biological properties of the cultured cardiomyocytes, among others.

[0005] Accordingly, there remain a number of challenges with respect to the production of in vitro models of cells according to an organized network.SUMMARY

[0006] In accordance with an aspect, there is provided a system for preparing a three- dimensional pattern in a cell culture well to enable an aligned growth of cultured cells, the system comprising: a patterning layer holder, the patterning layer holder comprising: an elongated body, the elongated body comprising: a patterning layer engaging portion provided in a distal region of the elongated body and sized for insertion into the cell culture well; and a peripheral wall having an outer peripheral wall surface, the peripheral wall defining a patterning layer cavity within the patterning layer engaging portion, the patterning layer cavity being configured to house therein at least a proximal portion of a patterning layer, the peripheral wall further defining a longitudinally-extending through-hole extending from the patterning layer cavity to a proximal end of the elongated body, the longitudinally-extending through-hole being configured to receive therein a patterning layer-creating material in a pre-cast form to subsequently cast the patterning layer in situ within the patterning layer cavity.

[0007] In some implementations, the patterning layer engaging portion is a tapering patterning layer engaging portion that tapers inwardly to define a patterning layer engaging portion gap between a peripheral wall inner surface of the cell culture well and the outer peripheral wall surface that is larger than at the proximal end of the elongated body.

[0008] In some implementations, the peripheral wall of the elongated body further defines a lateral opening located in the patterning layer engaging portion to provide access to the patterning layer cavity.

[0009] In some implementations, the elongated body is a cylindrical elongated body.

[0010] In some implementations, the patterning layer holder further comprises a top platform provided at the proximal end of the elongated body, the top platform defining acasting hole in fluid communication with the longitudinally-extending through-hole of the patterning layer holder.

[0011] In some implementations, the top platform comprises a cell culture well abutting wall configured to abut a top rim of the cell culture well.

[0012] In some implementations, the top platform is a circular top platform.

[0013] In some implementations, the top platform is a polygonal top platform.

[0014] In some implementations, the polygonal top platform is shaped as a rectangular prism, a pentagonal prism, a hexagonal prism, a heptagonal prism, or an octagonal prism.

[0015] In some implementations, the top platform comprises at least one manipulation instrument receiving cavity defined in a top surface of the top platform to enable manipulation of the patterning layer holder by a manipulation instrument.

[0016] In some implementations, the at least one manipulation instrument receiving cavity comprises a pair of manipulation instrument receiving cavities.

[0017] In some implementations, the at least one manipulation instrument receiving cavity is D-shaped.

[0018] In some implementations, the top platform comprises in indicia indicative of a pattern type provided on a top surface of the top platform.

[0019] In some implementations, the top platform comprises in indicia indicative of an orientation of the patterning layer, the indicia being provided on a top surface of the top platform.

[0020] In some implementations, the patterning layer holder comprises first and second patterning layer holders provided adjacent to each other, the first and second patterning layer holders having a common top platform and being provided at a distance from each other that enables insertion of the patterning layer engaging portion of each of the first and second patterning layer holders in a corresponding cell culture well.

[0021] In some implementations, the distance extends from a first central axis of the first patterning layer holder to the second central axis of the second patterning layer holder and corresponds to a well-spacing distance measured between a first well central axis and a second well central axis.

[0022] In some implementations, patterning layer holder comprises a plurality of patterning layer holders corresponding to a number of cell culture wells of a cell culture plate with which the plurality of patterning layer holders is intended to be used.

[0023] In some implementations, the cell culture plate includes 6, 12, 24, 48 or 96 cell culture wells.

[0024] In some implementations, the plurality of patterning layer holders is a monolithic array of patterning layer holders.

[0025] In some implementations, the system further comprises an electrode provided in at least one cell culture well of the cell culture wells of the cell culture plate.

[0026] In some implementations, the cell culture plate is a high-throughput electrophysiology cell culture plate that includes a microelectrode array (MEA) plate.

[0027] In some implementations, the system further comprises the patterning layer.

[0028] In some implementations, the patterning layer comprises protrusions defining a pattern-creating material receiving recess configured to receive a pattern-creating material therein such that the pattern-creating material received within the pattern-creating material receiving recess subsequently creates the three-dimensional pattern once deposited onto a pattern-receiving surface of the cell culture well.

[0029] In some implementations, the patterning layer-creating material from which is made the patterning layer comprises a polymeric material.

[0030] In some implementations, the polymeric material is an elastomeric material.

[0031] In some implementations, the elastomeric material comprises polydimethylsiloxane.

[0032] In some implementations, the patterning layer is a removable patterning layer.

[0033] In accordance with another aspect, there is provided a system for preparing a three-dimensional pattern in a cell culture well to enable an aligned growth of cultured cells, the system comprising: a patterning layer holder, the patterning layer holder comprising: an elongated body, the elongated body comprising: a patterning layer engaging portion provided at a distal end of the elongated body and being sized for insertion into the cell culture well; and a peripheral wall having an outer peripheral wall surface, the peripheral wall defining a patterning layer cavity within the patterning layer engaging portion; and a patterning layer having at least a proximal portion receivable within the patterning layer cavity, the patterning layer having a distal portion comprising protrusions defining a pattern-creating material receiving recess together provided according to a predetermined spatial distribution specific to the three-dimensional pattern, the patterning layer being configured to receive a pattern-creating material within the pattern-creating material receiving recess.

[0034] In some implementations, the patterning layer is configured such that the patterncreating material remains within the pattern-creating material receiving recess once cast and following retrieval of the patterning layer from the cell culture well to create the three- dimensional pattern on a pattern-receiving surface of the cell culture well.

[0035] In some implementations, the pattern-creating material received within the pattern-creating material receiving recess is transferable onto an additional patternreceiving surface of an additional cell culture well to create an additional three-dimensional pattern.

[0036] In some implementations, the pattern-creating material receiving recess comprises parallel furrows.

[0037] In some implementations, the protrusions are provided as staggered protrusions.

[0038] In some implementations, the protrusions are provided as a grid.

[0039] In some implementations, the peripheral wall further defines a longitudinally- extending through-hole extending from the patterning layer cavity to a proximal end of the elongated body, the longitudinally-extending through-hole being configured to receive therein a patterning layer-creating material in a pre-cast form to subsequently cast the patterning layer in situ within the patterning layer engaging portion.

[0040] In some implementations, the patterning layer-creating material from which is made the patterning layer comprises a polymeric material.

[0041] In some implementations, the polymeric material is an elastomeric material.

[0042] In some implementations, the elastomeric material comprises polydimethylsiloxane.

[0043] In some implementations, the patterning layer is a removable patterning layer.

[0044] In some implementations, the patterning layer holder comprises first and second patterning layer holders provided adjacent to each other, the first and second patterning layer holders having a common top platform and being provided at a distance from each other that enables insertion of the patterning layer engaging portion of each of the first and second patterning layer holders in a corresponding cell culture well.

[0045] In some implementations, the distance extends from a first central axis of the first patterning layer holder to the second central axis of the second patterning layer holder and corresponds to a well-spacing distance measured between a first well central axis and a second well central axis.

[0046] In some implementations, patterning layer holder comprises a plurality of patterning layer holders corresponding to a number of cell culture wells of a cell culture plate with which the plurality of patterning layer holders is intended to be used.

[0047] In some implementations, the cell culture plate includes 6, 12, 24, 48, or 96 cell culture wells.

[0048] In some implementations, the plurality of patterning layer holders is a monolithic array of patterning layer holders.

[0049] In some implementations, the system further comprises an electrode provided in at least one cell culture well of the cell culture wells of the cell culture plate.

[0050] In some implementations, the cell culture plate comprises the cell culture plate is a high-throughput electrophysiology cell culture plate that includes a microelectrode array (MEA) plate.

[0051] In accordance with another aspect, there is provided a system for preparing three-dimensional patterns in cell culture wells of a cell culture plate to enable an aligned growth of cultured cells, the system comprising: a plurality of patterning layer holders, each patterning layer holder of the patterning layer holders comprising: an elongated body, the elongated body comprising: a patterning layer engaging portion provided at a distal end of the elongated body and sized for insertion into the cell culture well; and a peripheral wall having an outer peripheral wall surface, the peripheral wall defining a patterning layer receiving cavity within the patterning layer engaging portion, the patterning layer receiving cavity being configured to receive therein at least a proximal portion of a patterning layer; a common top platform provided at respective proximal ends of the elongated bodies of the plurality of patterning layer holders such that the elongated bodies of the plurality of patterning layer holders extend downwardly from the common top platform.

[0052] In some implementations, the peripheral wall further defines a longitudinally- extending through-hole extending from the patterning layer cavity to a proximal end of the elongated body, the longitudinally-extending through-hole being configured to receive therein a patterning layer-creating material in a pre-cast form to subsequently cast a patterning layer in situ within the patterning layer engaging portion.

[0053] In some implementations, the top platform defines a plurality of casting holes in fluid communication with a corresponding longitudinally-extending through-hole of the plurality of patterning layer holders.

[0054] In some implementations, the patterning layer engaging portion is a tapering patterning layer engaging portion that tapers inwardly to define a patterning layer engaging portion gap between a peripheral wall inner surface of the cell culture well and the outer peripheral wall surface that is larger than at the proximal end of the elongated body.

[0055] In some implementations, the peripheral wall of the elongated body further defines a lateral opening located in the patterning layer engaging portion to provide access to the patterning layer cavity.

[0056] In some implementations, the elongated body is a cylindrical elongated body.

[0057] In some implementations, the top platform comprises a cell culture well abutting wall configured to abut a top rim of the cell culture wells.

[0058] In some implementations, the cell culture plate includes 6, 12, 24, 48, or 96 cell culture wells.

[0059] In some implementations, the plurality of patterning layer holders and the common top platform is a monolithic array of patterning layer holders.

[0060] In some implementations, the system further comprises an electrode provided in at least one cell culture well of the cell culture wells of the cell culture plate.

[0061] In some implementations, the cell culture plate is a high-throughput electrophysiology cell culture plate that includes a microelectrode array (MEA) plate.

[0062] In some implementations, the system further comprises the patterning layer.

[0063] In some implementations, the patterning layer comprises protrusions defining a pattern-creating material receiving recess configured to receive a pattern-creating material therein such that the pattern-creating material received within the pattern-creating material receiving recess subsequently creates the three-dimensional pattern once deposited onto the pattern-receiving surface of the cell culture well.

[0064] In some implementations, the patterning layer-creating material from which is made the patterning layer comprises a polymeric material.

[0065] In some implementations, the polymeric material is an elastomeric material.

[0066] In some implementations, the elastomeric material comprises polydimethylsiloxane.

[0067] In some implementations, the patterning layer is a removable patterning layer.

[0068] In accordance with another aspect, there is provided a system for applying a pattern-creating material onto a pattern-receiving surface, the system comprising: a patterning layer having a distal portion comprising protrusions provided according to a predetermined spatial distribution specific to a three-dimensional pattern and defining a pattern-creating material receiving recess configured to receive therein a pattern-creating material in a pre-cast form when the patterning layer is at a casting location; wherein the distal portion is configured to retain the pattern-creating material within the pattern-creating material receiving recess once the pattern-creating material is in a cast form and when the patterning layer is moved from the casting location to a culture location where the pattern-receiving surface is located.

[0069] In some implementations, the patterning layer is configured such that the protrusions extend upwardly to receive the pattern-creating material from above.

[0070] In some implementations, the patterning layer is configured such that the protrusions extend downwardly, and the distal portion is configured to be inserted into a reservoir containing the pattern-creating material.

[0071] In some implementations, the reservoir is surface-treated to be hydrophobic.

[0072] In some implementations, the pattern-receiving surface is hydrophobic.

[0073] In some implementations, the pattern-receiving surface is hydrophilic.

[0074] In some implementations, the pattern-creating material comprises a three- dimensional network of crosslinked hydrophilic polymers.

[0075] In some implementations, the protrusions have a cross-section selected between a polygonal cross-section, a substantially circular cross-section and an elliptic crosssection.

[0076] In some implementations, the pattern-creating material is configured to receive cells therein.

[0077] In some implementations, the cells comprising one or more of muscle cells, osteoclasts, fibroblasts, myoblasts, and neurons.

[0078] In some implementations, the protrusions provide a physical support for interacting with the cells.

[0079] In some implementations, the cells are muscle cells and the protrusions comprise a first protrusion and a second protrusion for the muscle cells to grow in between.

[0080] In some implementations, the pattern-receiving surface is part of a receptacle.

[0081] In some implementations, the receptacle is a cell culture dish or a cell culture well.

[0082] In some implementations, the receptacle is configured to receive an additional cell culture therein.

[0083] In some implementations, the receptacle is configured to receive a cell culture therein and to receive the pattern-creating material once in the cast form.

[0084] In some implementations, the cell culture well is configured to receive a cell culture therein and to receive the patterning later and pattern-creating material once in the cast form.

[0085] In some implementations, the cell culture comprises neurons.

[0086] In some implementations, the system further comprises a patterning layer holder, the patterning layer holder comprising:an elongated body, the elongated body comprising: a patterning layer engaging portion provided at a distal end of the elongated body; and a peripheral wall having an outer peripheral wall surface, the peripheral wall defining a patterning layer cavity within the patterning layer engaging portion configured to receive at least a proximal portion of the patterning layer therein.

[0087] In accordance with another aspect, there is provided a method for preparing first and second three-dimensional patterns on first and second pattern-receiving surfaces of first and second cell culture wells respectively to enable an aligned growth of cultured cells, the method comprising: introducing a pattern-creating material in a pre-cast form into the first cell culture well; inserting a distal portion of a patterning layer into the pattern-creating material, the patterning layer comprising protrusions defining a pattern-creating material receiving recess provided according to a predetermined spatial distribution specific to the first and second three-dimensional patterns; incubating the pattern-creating material for a casting time period sufficient to transition the pattern-creating material from the pre-cast form to a cast form; retrieving the patterning layer combined with the pattern-creating material received within the pattern-creating material receiving recess from the first cell culture well, thereby creating the first three-dimensional pattern on the first pattern-receiving surface; inserting the patterning layer combined with the pattern-creating material received within the pattern-creating material receiving recess into the second cell culture well; depositing the pattern-creating material received within the pattern-creating material receiving recess onto the second pattern-receiving surface of the second cell culture well, thereby creating the second three-dimensional pattern on the second patternreceiving surface; andretrieving the patterning layer from the second cell culture well.

[0088] In accordance with another implementation, there is provided a method for preparing first and second three-dimensional patterns on first and second patternreceiving surfaces of first and second cell culture wells respectively to enable an aligned growth of cultured cells, the method comprising: introducing a pattern-creating material in a pre-cast form into the first cell culture well; incubating the pattern-creating material for a casting time period sufficient to transition the pattern-creating material from the pre-cast form to a cast form; inserting a distal portion of a patterning layer into the pattern-creating material, the patterning layer comprising protrusions defining a pattern-creating material receiving recess provided according to a predetermined spatial distribution specific to the first three-dimensional patterns; retrieving the patterning layer combined with the pattern-creating material received within the pattern-creating material receiving recess from the first cell culture well, thereby creating the first three-dimensional pattern on the first pattern-receiving surface; inserting the patterning layer combined with the pattern-creating material received within the pattern-creating material receiving recess into the second cell culture well; depositing the pattern-creating material received within the pattern-creating material receiving recess onto the second pattern-receiving surface of the second cell culture well, thereby creating the second three-dimensional pattern on the second patternreceiving surface; and retrieving the patterning layer from the second cell culture well.

[0089] In some implementations, the first and second three-dimensional patterns correspond to first and second sets of three-dimensional patterns respectively.

[0090] In some implementations, a number of first three-dimensional patterns in the first set of three-dimensional patterns corresponds to a number of the first cell culture wells of a first cell culture plate.

[0091] In some implementations, the first cell culture plate includes 6, 12, 24, 48, or 96 cell culture wells.

[0092] In some implementations, a number of second three-dimensional patterns in the second set of three-dimensional patterns corresponds to a number of the second cell culture wells of a second cell culture plate.

[0093] In some implementations, the first and second cell culture plated includes the same number of cell culture wells.

[0094] In some implementations, the method further comprises providing an electrode in at least one cell culture well of the first and second cell culture wells.

[0095] In some implementations, at least one of the first and second cell culture plates comprises a microelectrode array plate.

[0096] In some implementations, the casting time period ranges from about 5 minutes to about 90 minutes.

[0097] In some implementations, incubating the pattern-creating material for the casting time period comprises applying a downward pressure onto the patterning layer during the casting time period.

[0098] In some implementations, the method further comprises rinsing the first three- dimensional pattern with a rinsing medium.

[0099] In some implementations, depositing the pattern-creating material received within the pattern-creating material receiving recess onto the pattern-receiving surface of the second cell culture well comprises applying a downward pressure onto the patterning layer.

[0100] In some implementations, the method further comprises rinsing the second three- dimensional pattern with a rinsing medium.

[0101] In accordance with another aspect, there is provided a method for preparing a three-dimensional pattern in a cell culture well of a cell culture plate, the method comprising: introducing a patterning layer-creating material into a longitudinally-extending through-hole extending of an elongated body of a patterning layer holder to create a patterning layer received within a patterning layer cavity of a patterning layer engaging portion of the elongated body, the patterning layer comprising protrusions defining a pattern-creating material receiving recess provided according to a predetermined spatial distribution specific to the three-dimensional pattern; introducing a pattern-creating material into the cell culture well; inserting a distal portion of the patterning layer into the pattern-creating material such that the pattern-creating material is received within the pattern-creating material receiving recess of the patterning layer; incubating the pattern-creating material for a casting time period sufficient to transition the pattern-creating material from the pre-cast form to a cast form; and retrieving the patterning layer combined with the pattern-creating material received within the pattern-creating material receiving recess from the cell culture well, thereby creating the three-dimensional pattern on a pattern-receiving surface of the cell culture well.

[0102] In some implementations, the method further comprises: inserting the patterning layer combined with the pattern-creating material received within the pattern-creating material receiving recess into an additional cell culture well of an additional cell culture plate; and depositing the pattern-creating material received within the pattern-creating material receiving recess onto an additional pattern-receiving surface of the additional cell culture well, thereby creating an additional three-dimensional pattern on the additional pattern-receiving surface.

[0103] In some implementations, the casting time period ranges from about 5 minutes to about 90 minutes.

[0104] In some implementations, incubating the pattern-creating material for the casting time period comprises applying a downward pressure onto the patterning layer during the casting time period.

[0105] In some implementations, depositing the pattern-creating material received within the pattern-creating material receiving recess onto the additional pattern-receiving surface of the additional cell culture well comprises applying a downward pressure onto the patterning layer.

[0106] In some implementations, the patterning layer holder comprises first and second patterning layer holders provided adjacent to each other, the first and second patterning layer holders having a common top platform and being provided at a distance from each other that enables insertion of the patterning layer engaging portion of each of the first and second patterning layer holders in a corresponding cell culture well.

[0107] In some implementations, patterning layer holder comprises a plurality of patterning layer holders corresponding to a number of cell culture wells of a cell culture plate with which the plurality of patterning layer holders is intended to be used.

[0108] In some implementations, the cell culture plate includes 6, 12, 24, 48, or 96 cell culture wells.

[0109] In some implementations, the patterning layer holders is a monolithic array of patterning layer holders.

[0110] In some implementations, the method further comprises providing an electrode in at least one cell culture well of the cell culture wells of the cell culture plate.

[0111] In some implementations, the cell culture plate comprises a microelectrode array plate.

[0112] In accordance with another aspect, there is provided a method for applying a pattern-creating material onto a pattern-receiving surface, the method comprising:contacting, at a casting location, the pattern-creating material in a pre-cast form with a distal portion of a patterning layer, the distal portion comprising protrusions provided according to a predetermined spatial distribution specific to a three- dimensional pattern and defining a pattern-creating material receiving recess; incubating the pattern-creating material for a casting time period sufficient to transition the pattern-creating material from the pre-cast form to a cast form; and transferring at least the pattern-creating material in the cast form to a culture location to apply the at least the pattern-creating material in the cast form onto the patternreceiving surface.

[0113] In some implementations, contacting the pattern-creating material in the pre-cast form with the distal portion of the patterning layer comprises pouring the pattern-creating material in the pre-cast form onto the distal portion of the patterning layer the patterning layer while the protrusions extend upwardly.

[0114] In some implementations, contacting the pattern-creating material in the pre-cast form with the distal portion of the patterning layer comprises inserting the distal portion of the patterning layer into a reservoir containing the pattern-creating material while the protrusions extend downwardly.

[0115] In some implementations, the reservoir is hydrophobic.

[0116] In some implementations, the pattern-receiving surface is hydrophobic.

[0117] In some implementations, the pattern-receiving surface is hydrophilic.

[0118] In some implementations, the pattern-creating material comprises a three- dimensional network of crosslinked hydrophilic polymers.

[0119] In some implementations, the protrusions have a cross-section selected between a polygonal cross-section, a substantially circular cross-section and an elliptic crosssection.

[0120] In some implementations, the pattern-creating material contains cells.

[0121] In some implementations, the cells comprising one or more of muscle cells, osteoclasts, fibroblasts, myoblasts, and neurons.

[0122] In some implementations, the protrusions provide a physical support for interacting with the cells.

[0123] In some implementations, the cells comprise muscle cells and the protrusions comprise a first protrusion and a second protrusion for the muscle cells to grow in between.

[0124] In some implementations, applying the at least the pattern-creating material in the cast form onto the pattern-receiving surface comprises applying the pattern-creating material in the cast form and the patterning layer onto the pattern-receiving surface in an upward orientation.

[0125] In some implementations, applying the at least the pattern-creating material in the cast form onto the pattern-receiving surface comprises applying the pattern-creating material in the cast form and the patterning layer onto the pattern-receiving surface in a downward orientation.

[0126] In some implementations, the method further comprises incubating the cells received in the pattern-creating material with an additional cell culture.

[0127] In some implementations, the pattern-receiving surface is part of a receptacle.

[0128] In some implementations, the receptacle is a cell culture dish or a cell culture well.

[0129] In some implementations, the receptacle is configured to receive an additional cell culture therein.

[0130] In some implementations, the method further comprises incubating the patterncreating material in the cast form with the additional cell culture received in the receptacle.

[0131] In some implementations, the method further comprises incubating the patterncreating material in the cast form combined with the patterning later with the additional cell culture received in the receptacle.

[0132] In some implementations, the additional cell culture comprises neurons.

[0133] In some implementations, at least a proximal portion of the patterning is engageable with a patterning layer holder, the patterning layer holder comprising: an elongated body, the elongated body comprising: a patterning layer engaging portion provided at a distal end of the elongated body; and a peripheral wall having an outer peripheral wall surface, the peripheral wall defining a patterning layer cavity within a patterning layer engaging portion configured to receive the at least a proximal portion of the patterning layer.BRIEF DESCRIPTION OF THE DRAWINGS

[0134] The attached figures illustrate various features, aspects and implementations of the technology described herein.

[0135] Fig 1 is a perspective exploded view of a patterning layer holder and a patterning layer, in accordance with an implementation.

[0136] Fig 2 is an upside-down perspective exploded view of the patterning layer holder and the patterning layer of Fig 1.

[0137] Fig 3 is a top view of the patterning layer holder and the patterning layer of Fig 1.

[0138] Fig 4 is a front cross-sectional view of the patterning layer holder and the patterning layer of Fig 1 , with the patterning layer being received in a patterning layer cavity of the patterning layer holder.

[0139] Fig 5 is a front cross-sectional view of the patterning layer holder of Fig 1 , without the patterning layer.

[0140] Fig 6 is a front cross-sectional view of the patterning layer holder and the patterning layer of Fig 1 , shown received within a cell culture well.

[0141] Fig 7 is a perspective cross-sectional view of the patterning layer holder and the patterning layer of Fig 4.

[0142] Fig 8 is a perspective exploded view of a patterning layer holder, a patterning layer and a patterning mold, in accordance with another implementation.

[0143] Fig 9 is a top view of the patterning layer holder of Fig 8.

[0144] Figs 10A to 10E are top views of examples of three-dimensional patterns.

[0145] Fig 11 is a perspective view of a patterning layer holder, in accordance with another implementation.

[0146] Fig 12 is a front cross-sectional view of the patterning layer holder of Fig 11.

[0147] Fig 13 is a perspective cross-sectional view of the patterning layer holder of Fig 11.

[0148] Fig 14 is a top perspective view of a cell culture plate with a microelectrode array plate, in accordance with an implementation.

[0149] Fig 15 is an enlarged view of a portion of the cell culture plate of Fig 14.

[0150] Fig 16 is a front perspective view of a patterning system that includes a common top platform and four patterning layer holders.

[0151] Fig 17 is a top view of the patterning system of Fig 16.

[0152] Fig 18 is a front perspective view of a patterning system that includes a common top platform and six patterning layer holders.

[0153] Fig 19 is a top view of the patterning system of Fig 18.

[0154] Fig 20 is a front perspective view of a patterning system that includes a common top platform and twelve patterning layer holders.

[0155] Fig 21 is a top view of the patterning system of Fig 20.

[0156] Fig 22 is a front perspective view of a patterning system that includes a common top platform and sixteen patterning layer holders.

[0157] Fig 23 is a top view of the patterning system of Fig 22.

[0158] Figs 24A to 24H are schematic representations illustrating method Steps 200 to 214.

[0159] Figs 25A to 25D are additional schematic representations illustrating method Steps 200 to 206.

[0160] Figs 26A to 26D are additional schematic representations illustrating method Steps 208 to 214.

[0161] Fig 27A to 27H are schematic representations illustrating method Steps 400 to 414.

[0162] Fig 28 is a perspective view of a schematic representation of a pattern-creating material in a pre-cast form being added onto a patterning layer, in accordance with an implementation.

[0163] Fig 29 is a side view of a schematic representation of the pattern-creating material and the patterning layer of Fig 28, in the pre-cast form.

[0164] Fig 30 is a side view of a schematic representation of a pattern-creating material in a cast form.

[0165] Fig 31 is a side view of a schematic representation of a pattern-creating material in a cast form, a patterning layer and a patterning layer holder shown engaged and in an upward orientation.

[0166] Fig 32 is a side view of a schematic representation of a pattern-creating material in a cast form and a patterning layer shown in an upward orientation in a receptacle.

[0167] Fig 33 is a side view of a schematic representation of a pattern-creating material in a cast form, a patterning layer and a patterning layer holder shown engaged and in a downward orientation in a receptacle.

[0168] Fig 34 is a side view of a schematic representation of a pattern-creating material in a cast form transferred into a receptacle.

[0169] Fig 35 is a side view of a schematic representation of a pattern-creating material in a cast form, a patterning layer and a patterning layer holder shown engaged and in a downward orientation in a receptacle.DETAILED DESCRIPTION

[0170] Techniques described herein relate to systems, devices, and methods for preparing three-dimensional patterns in a cell culture well of a cell culture plate in an efficient and reproducible manner. The three-dimensional patterns can be useful for culturing various types of cells that can benefit from being cultured according to an organized network. Examples of such cells include cardiomyocytes, neurons, myoblasts, and induced pluripotent stem cells during certain differentiation techniques. For example, culturing cardiomyocytes according to an organized network can enable obtaining an aligned, and in some instance a coordinated, growth of the cardiomyocytes, which can enable the cultured cardiomyocytes to better mimic the in vivo functionalities of cardiac tissues, among other benefits.

[0171] The preparation of the three-dimensional patterns in cell culture wells of a cell culture plate as described herein can be compatible with the industry standard High Throughput Screening (HTS) format, and can be used for a wide range of cellular assays including contractile mechanical output, electrical activity, compound screening, compound discovery, safety and efficacy testing, etc.

[0172] The three-dimensional patterns can be obtained using a patterning layer holder as described herein. The patterning layer holder can be provided as a single patterning layer holder, or a plurality of patterning layer holders can be provided, for instance as an array of patterning layer holders (e.g., a monolithic array of patterning layer holders), to form a patterning system. The number of patterning layer holders can correspond to the number of cell culture wells of the cell culture plate into which the three-dimensional patterns are intended to be prepared. When the number of patterning layer holders corresponds to the number of cell culture wells of the cell culture plate into which the three- dimensional patterns are intended to be prepared, a corresponding number of three-dimensional patterns can be prepared simultaneously, which can greatly reduce the time necessary for preparing multiple of three-dimensional patterns. In addition, this technique can enable preparing a plurality of three-dimensional patterns that are aligned with each other.

[0173] Each patterning layer holder includes an elongated body configured for interacting with a patterning layer. The elongated body includes a patterning layer engaging portion provided at a distal end of the elongated body, the patterning layer engaging portion being sized for insertion into the cell culture well. The elongated body includes a peripheral wall defining a patterning layer cavity within the patterning layer engaging portion. The patterning layer cavity is configured to house therein at least a proximal portion of the patterning layer. The patterning layer includes a distal portion comprising protrusions provided according to a predetermined spatial distribution specific to the three-dimensional pattern that is desired to be obtained, the protrusions defining a pattern-creating material receiving recess therebetween. Although a cell culture well is used as an example of receptacle to receive a three-dimensional pattern, it is to be understood that in alternative implementations, any type of receptacle can also be suitable.

[0174] In some implementations, the peripheral wall can further define a longitudinally- extending through-hole extending from the distal end to a proximal end of the elongated body. The longitudinally-extending through-hole can be configured to receive therein a patterning layer-creating material in a pre-cast form to subsequently cast a patterning layer in situ within the patterning layer cavity. The resulting patterning layer can then be retained within the patterning layer cavity of the patterning layer engaging portion. Thus, the patterning layer holder can be used to accomplish dual actions to further improve the efficiency of the preparation of multiple of three-dimensional patterns, by enabling the creation of the patterning layer in situ and then the insertion of the patterning layer into a cell culture well to create the desired three-dimensional pattern in the cell culture well. Alternatively, the longitudinally-extending through-hole may be omitted, and the patterning layer can be provided by other means, / .e., without having been created via the longitudinally-extending through-hole.

[0175] The patterning layer is configured to receive a pattern-creating material within the pattern-creating material receiving recess. This can be achieved by introducing, or pouring, a pattern-creating material in a pre-cast form into a cell culture well, or another type of receptacle, and then incubating the pattern-creating material for a casting time period sufficient to transition the pattern-creating material from the pre-cast form to a cast form. A distal portion of the patterning layer can be inserted into the pattern-creating material, either prior to, during, or after the casting time period. When the patterning layer combined with the pattern-creating material received within the pattern-creating material receiving recess is subsequently retrieved from the cell culture well, a three-dimensional pattern is created on the pattern-receiving surface of the cell culture well (or another type of receptacle). Alternatively, the pattern-creating material in pre-cast form can be added directly onto the patterning layer, for instance if the patterning layer is provided in an upward configuration.

[0176] If it is desired to create an additional three-dimensional pattern in an additional cell culture well, which can be in the same cell culture plate or be in another cell culture plate, the patterning layer combined with the pattern-creating material received within the pattern-creating material receiving recess can be inserted into the additional cell culture well, and the pattern-creating material received within the pattern-creating material receiving recess can be deposited onto the additional pattern-receiving surface of the additional cell culture well, thereby creating the additional three-dimensional pattern on the additional pattern-receiving surface of the additional cell culture well.

[0177] Thus, the combination of the patterning layer holder and the patterning layer as described herein can further contribute to improving the efficiency of the preparation of multiple of three-dimensional patterns by enabling the creating of two three-dimensional patterns per single dipping of the distal portion of the patterning layer into the patterncreating material.

[0178] It will be appreciated that positional descriptions such as “above”, “below”, “left”, “right”, “inwardly”, “outwardly”, “distal”, “proximal” and the like used in the present description should, unless otherwise indicated, be taken in the context of the figures and should not be considered limiting. The term “outwardly” is intended to refer to a feature that extends toward an exterior side of a reference axis. The term “inwardly” is intendedto refer to a feature that extends toward an interior side of a reference axis. The term “distal” is meant to refer to a feature of the system that is provided closest to a patternreceiving surface of a cell culture well, when the patterning layer holder is placed in an upright position. The term “proximal” is meant to refer to a feature of the system that is provided farthest to a pattern-receiving surface of a cell culture well, when the patterning layer holder is placed in an upright position.

[0179] Various implementations of the patterning layer holder and associated patterning system will now be described in greater detail.Description of the patterning layer holder

[0180] With reference to Figs 1 to 9, an implementation of a patterning layer holder 10 is shown. The patterning layer holder 10 includes an elongated body 12 having a cylindrical shape. The elongated body 12 includes a patterning layer engaging portion 14 provided in a distal region 16 of the elongated body 12. In the implementation shown, the patterning layer engaging portion 14 is sized for insertion into a cell culture well of a cell culture plate. It is to be understood that in other implementations, the patterning layer engaging portion 14 can have other dimensions depending on the intended use of the patterning layer holder 10. The size of the patterning layer engaging portion 14 can thus be adapted to the size, and in particular the height and diameter, of the cell culture well into which patterning layer engaging portion 14 is tended to be inserted, which can vary for cell culture plates having for instance 6, 12, 24, 48, or 96 cell culture wells.

[0181] The elongated body 12 includes a peripheral wall 18. The peripheral wall 18 defines a patterning layer cavity 20 within the patterning layer engaging portion 14. The patterning layer cavity 20 is configured to house therein at least a proximal portion 22 of a patterning layer 24. The peripheral wall 18 includes a patterning layer abutting wall 30 and a patterning layer cavity sidewall 32. More details regarding the configuration of the patterning layer 24 are provided below.

[0182] In the implementation shown in Figs 1 to 9, the peripheral wall 18 further defines a longitudinally-extending through-hole 26 extending from the patterning layer engaging portion 14 to a proximal end 28 of the elongated body 12. The longitudinally-extending through-hole 26 is in fluid communication with the patterning layer cavity 20 of thepatterning layer engaging portion 14. The longitudinally-extending through-hole 26, when present, is configured to receive therein a patterning layer-creating material in a pre-cast form to subsequently cast a patterning layer 24 in situ within the patterning layer cavity 20. In order to do so, the patterning layer-creating material can be introduced via (e.g., poured into) the longitudinally-extending through-hole 26 until it reaches the patterning layer cavity 20 while a distal end 34 of the elongated body 12 is in contact with a patterning mold 36, as shown in Fig 8. In some implementations, the patterning mold 36 can also be referred to as a “wafer”. The patterning layer-creating material is allowed to cast for a given period of time (e.g., under curing conditions), and then the patterning layer holder 10 is retrieved while the proximal portion 22 of the patterning layer 24 remains within the patterning layer cavity 20. This results in the configuration of the combination of the patterning layer holder 10 and the patterning layer 24 assembled as shown in Fig 4.

[0183] The combination of the patterning layer abutting wall 30 and the patterning layer cavity sidewall 32 defined by the peripheral wall 18 of the elongated body 12 along with the patterning mold top surface 38 of the patterning mold 36 thus form an enclosed mold into which the patterning layer-creating material can be introduced via the longitudinally- extending through-hole 26 to create the patterning layer 24.

[0184] Still referring to Figs 1 to 9, the patterning layer engaging portion 14 provided in the distal region 16 of the elongated body 12 is exemplified as a tapering patterning layer engaging portion that tapers inwardly to define a patterning layer engaging portion gap between a peripheral wall inner surface of the cell culture well and the outer peripheral wall surface of the elongated body 12 that is larger than at the proximal end 28 of the elongated body 12. In some implementations, the tapering patterning layer engaging portion can facilitate the insertion of the elongated body 12 of the patterning layer holder 10 into the cell culture well of the cell culture plate, while the remainder of the elongated body 12 can have a slightly larger diameter, or width, that can contribute to stabilizing the elongated body 12 into the cell culture well of the cell culture plate by limiting the side movement of the elongated body 12 relative to the peripheral wall inner surface of the cell culture well. In addition, a patterning layer engaging portion 14 that is provided as a tapering patterning layer engaging portion can facilitate the displacement of fluids and air when the patterning layer holder 10 is used to prepare three-dimensional patterns within a cell culture well of a cell culture layer.

[0185] In that regard, it is to be noted that although the elongated body 12 of the patterning layer holder 10 shown in Figs 1 to 9 is exemplified as having a substantially cylindrical shape, in alternative implementations, the elongated body 12 can have another shape and take the form for instance of polygonal prism having lateral sides. In some implementations, the cylindrical shape of the elongated body 12 can facilitate the interaction of the elongated body with the peripheral wall inner surface of the cell culture well, but a polygonal prism, or any other geometrical shape, can also be suitable, with the lateral edges of the polygon being engageable with the peripheral wall inner surface of the cell culture well.

[0186] In some implementations and as shown more particularly to Figs 1 , 2 and 8, the peripheral wall 18 of the elongated body 12 further defines a lateral opening 40 located in the patterning layer engaging portion 14 to provide access to the patterning layer cavity 20. In the implementation shown, the peripheral wall 18 of the elongated body 12 defines two lateral openings 40 that are diametrically opposed to each other. In alternative implementations, there can be more than two lateral openings or there can be a single lateral opening, and the lateral openings can be located at other locations than those illustrated. The lateral opening 40 can facilitate accessing the patterning layer 24 when the patterning layer 24 is located in the patterning layer cavity 20. For instance, the lateral opening 40 can enable forceps to enter into the patterning layer cavity 20 to contact the patterning layer 24, for instance to remove the patterning layer 24 from the patterning layer cavity 20, or to insert the patterning layer 24 into the patterning layer cavity 20. In the implementation shown, the lateral openings 40 have a substantially rectangular shape. It is to be understood that in other implementations, the lateral openings 40 can have a different shape. For instance, the lateral openings 40 can be shaped as a notch, can have a width similar to a height (and thus be shaped as a square), etc.

[0187] Still referring to Figs 1 to 9, the patterning layer holder 10 further includes a top platform 42 provided at the proximal end 44 of the elongated body 12. The top platform 42 defines a casting hole 46 in fluid communication with the longitudinally-extending through-hole 26 of the elongated body 12 of the patterning layer holder 10. The top platform 42 can include a cell culture well abutting wall 48 configured to abut a top rim 50 of a cell culture well 52 (or another type of receptacle), as shown in Fig 6. In the implementation shown, the top platform 42 has a polygonal shape. More particularly andas shown in Fig 3, the top platform 42 is shaped as a hexagonal prism. In alternative implementations, the top platform 42 can be shaped as a rectangular prism, a pentagonal prism, a heptagonal prism, an octagonal prism, etc. In yet other implementations, the top platform 42 can be circular or oval. It is to be understood that the top platform 42 can have any suitable configuration and shape, which are not limited to the mentioned examples.

[0188] The top platform 42 can include at least one manipulation instrument receiving cavity 54 defined in a top surface 56 of the top platform 42 to enable manipulation of the patterning layer holder 10 by a manipulation instrument. In the implementation shown in Figs 1 to 7, the top platform 42 includes four manipulation instrument receiving cavities 54, provided as two pairs of diametrically opposed manipulation instrument receiving cavities 54. In alternative implementations and as shown in Figs 8 and 9 for instance, the top platform 42 can include two manipulation instrument receiving cavities 54 provided as a pair of diametrically opposed manipulation instrument receiving cavities 54. In the implementations shown, the manipulation instrument receiving cavities 54 are exemplified as being D-shaped, with the straight line of the “D” being oriented toward a central axis 58 of the longitudinally-extending through-hole 26. The manipulation instrument receiving cavities 54 are configured to enable the patterning layer holder 10 to be manipulated by a user, for instance using forceps, in the context of sterile practices. The patterning layer holder 10 can thus be manipulated by a user without having to grab the top platform 42 by hand. It is to be understood that other configurations of the manipulation instrument receiving cavity 54, including other shapes, are also possible and within the scope of the present description. In some implementations, the manipulation instrument receiving cavity 54 can be omitted.

[0189] Referring to Fig 9, the top surface 56 of the top platform 42 further includes an indicium 60. In the implementation shown, two indicia 60 shaped as the number “1” are illustrated. In general, the indicia chosen can be indicative of a pattern type of the patterning layer 24 and / or an orientation of the patterning layer 24. Any type of indicia can be selected for a corresponding category of information associated to the patterning layer holder as desired by the user. For instance, although a number is exemplified in Fig 9, the indicia 60 can be a letter or a distinct shape, or any other type of recognizable indicator that is linked to a given information about the patterning layer 24 or the patterning layer holder 10.Description of the patterning layer

[0190] Referring more particularly to Figs 1 , 2, 4 and 6 to 8 and as mentioned above, the elongated body 12 includes a peripheral wall 18 defining a patterning layer cavity 20 within the patterning layer engaging portion 14. The patterning layer cavity 20 is configured to house therein at least a proximal portion 22 of a patterning layer 24. The peripheral wall 18 includes a patterning layer abutting wall 30 and a patterning layer cavity sidewall 32. When the longitudinally-extending through-hole 26 is present in the elongated body 12 and used to receive therein a patterning layer-creating material in a pre-cast form to subsequently cast a patterning layer 24 in situ within the patterning layer cavity 20, the combination of the patterning layer abutting wall 30, the patterning layer cavity sidewall 32 and the patterning mold top surface 38 of the patterning mold 36 forms an enclosed mold into which the patterning layer-creating material can be introduced via the longitudinally- extending through-hole 26 to create the patterning layer 24. The patterning mold 36 can be any type of patterning mold having a desired pattern that will subsequently be used to produce the patterning layer 24 and then the three-dimensional pattern in the cell culture well of the cell culture plate. As shown in Figs 4, 6, and 7, the proximal portion 22 of the patterning layer 24 is housed in the patterning layer cavity 20, such that a distal portion 62 of the patterning layer 24 remains outside of the elongated body 12 to enable contact with a pattern-creating material present at the bottom of the cell culture well 52.

[0191] The distal portion 62 of the patterning layer 24 includes protrusions 66 defining a pattern-creating material receiving recess 64, the protrusions 66 being provided according to a predetermined spatial distribution specific to the three-dimensional pattern that it is desired and / or to a given use of the patterning later. The patterning layer 24 can be obtainable following the casting of the patterning layer-creating material against the patterning mold 36, or can be obtained according to other means, for instance if the longitudinally-extending through-hole 26 is not present in the elongated body 12. In some implementations, the patterning layer 24 is configured to receive a pattern-creating material within the pattern-creating material receiving recess 64, such that the patterncreating material remains within the pattern-creating material receiving recess 64 once cast and following the retrieval of the patterning layer 24 from the cell culture well 52, to create a three-dimensional pattern on a pattern-receiving surface 68 of the cell culture well 52. The pattern-creating material received within the pattern-creating material receivingrecess 64 can be further transferable onto an additional pattern-receiving surface 68 of an additional cell culture well, or any other type of pattern-receiving surface, to create an additional three-dimensional pattern. In some implementations, the patterning layer 24 is configured to receive a pattern-creating material within the pattern-creating material receiving recess 64, such that the pattern-creating material remains within the patterncreating material receiving recess 64 once in a cast form, and the retrieval of the patterning layer 24 can be omitted. In such implementations, maintaining the pattern-creating material in the cast form within the pattern-creating material receiving recess 64 can enable the protrusions 66 to play a role of a physical support for interacting with certain given types of cells. Furthermore, in some implementations, the additional patternreceiving surface of an additional cell culture well (or another type of pattern-receiving surface and / or another type of receptacle) may already include another cell culture, for instance cells cultured in a polymerizing solution, such as a hydrogel. These aspects and the method for obtaining the three-dimensional pattern(s) will be described in more detail below.

[0192] The pattern-creating material can be for instance a polymerizing solution, PDMS, any soft polymer such as silicone-based polymers, rubber materials, biological materials including for instance gelatine, agarose, collagen, a gel solution comprising Matrigel®, Geltrex®, fibrin, collagen, or other types of basement membrane matrix or extracellular membrane matrix (ECM), etc. In some implementations, the pattern-creating material can include a soft material formed by a three-dimensional network of crosslinked hydrophilic polymers capable of expending, or swelling, in a solvent such as an aqueous solvent, and retaining water or another fluid. An example of three-dimensional network of crosslinked hydrophilic polymers is a hydrogel (e.g., physical hydrogels and chemical hydrogels). Various types of hydrogels can be used, depending on the desired stimulus (e.g., temperature, electric and magnetic fields, solvent composition, light intensity, and pressure, while the chemical or biochemical stimuli include pH, ions, and specific chemical compositions), that will subsequently induce the volume phase transition or gel-sol phase transition of the hydrogel. In some implementations, the pattern-creating material can include one or more cell types. The pattern-creating material can itself include the one or more cell types, / .e., the pattern-creating material can include the one or more cell types already seeded therein. It is to be understood that the cells can also be seeded in a cell culture well or other receptacle, and the polymerizing solution, such as a hydrogel, can beadded afterward onto the cells. Examples of cells that can be present in the patterncreating material or seeded into the cell culture well can include for instance fibroblasts, myoblasts, smooth muscle cells, skeletal muscle cells, neurons, etc. It is to be understood that any type of cells can be suitable in accordance with the application chosen and the intended use.

[0193] In some implementations, the surface of the patterning layer 24 that includes the pattern-creating material receiving recess 64 and the protrusions 66, / .e., the patterncreating surface, can be surface-treated to provide hydrophilic or hydrophobic properties to the surface of the pattering layer 24 receiving the pattern-creating material. For instance, in some implementations, the surface of the patterning layer 24 that includes the pattern-creating material receiving recess 64 and the protrusions 66 can be surface- treated to be hydrophilic such that when the patterning layer 24 is displaced, the patterncreating material is released from the pattern-creating material receiving recess 64 and protrusions 66, for instance as shown in Step 212 of Fig 26C. In other implementations, the surface of the patterning layer 24 that includes the pattern-creating material receiving recess 64 and the protrusions 66 can be surface-treated to be hydrophobic such that when the patterning layer 24 is displaced, the pattern-creating material is retained within the pattern-creating material receiving recess 64 and protrusions 66, for instance as shown in Step 206 of Fig 25D. Such a configuration of the patterning layer 24 may be desirable for instance in situations where the protrusions 66 may also play a role of a physical support for interacting with certain given types of cells that may be present in the pattern-creating material. For instance, in some implementations, when the pattern-creating material includes muscle cells, the protrusions 66 (two or more) can act as pillars to facilitate the creation of a bundle of muscle cells therebetween. In some implementations, the surface of the patterning layer 24 that includes the pattern-creating material receiving recess 64 and the protrusions 66 can have properties enabling the retaining of the pattern-creating material within the pattern-creating material receiving recess 64 followed by the release of the pattern-creating material from the pattern-creating material receiving recess 64, as shown successively in Figs 25 and 26.

[0194] In some implementations, when the pattern-creating material includes cells for culture and regardless of whether the pattern-creating surface is surface-treated or not, the pattern-creating material retained onto the pattern-creating surface of the patterninglayer 24 can be configured to remain in position onto the pattern-creating surface and thus coupled with the patterning layer 24 within the cell culture well 52 (or another receptacle) such that cells can be cultured in proximity of one or more protrusions 66 of the patterning layer. This can be achieved for instance by using the patterning layer 24 in an upward orientation (e.g., with pouring the pattern-creating material onto the pattern-crating surface) or a downward orientation (e.g., resulting from the insertion of the distal portion 62 of the patterning layer 24 into a pool of pattern-creating material and retaining the pattern-creating material in contact with the pattern-creating surface while the patterning layer is retrieved from the cell culture well or other receptable into which the pool of patterncreating material is). In such implementations, the combination of the patterning layer and the culture of cells can be used as is, either in an upward orientation or a downward orientation, to continue further experiments onto the cultured cells and potentially additional cells as well (for instance if the combination of the patterning layer and the pattern-creating material in the cast form and containing the culture of cells is inserted in a downward orientation into a cell culture well or another receptable for continuing culturing cells).

[0195] The configuration and spatial distribution of the pattern-creating material receiving recess 64 and the protrusions 66 can be any configuration and spatial distribution that is desired to culture a given type of cells and / or to perform a given type of experiment. Examples of configurations and spatial distributions are illustrated in Figs 10A to 10E, for illustrative purposes only. For instance, in some implementations and as shown in Figs 10A to 10E, the pattern-creating material receiving recess 64 can be substantially parallel pattern-creating material receiving recess 64. In some implementations and as shown in Fig 10C, the protrusions 66 can be provided as a grid. In some implementations and as shown in Fig 10D, the protrusions 66 can be provided as staggered protrusions. In some implementations, the cross-section of the protrusions 66 can be any geometrical shape or polygonal shape (e.g., circular, triangular, rectangular, hexagonal, octagonal etc.). For instance, in Figs 10A to 10D, the cross-section of the protrusions is rectangular, and in Fig 10E, the cross-section of the protrusions 66 is substantially circular. The width of the pattern-creating material receiving recess 64 (measured between adjacent ones of the protrusions 66) and / or the protrusions 66 (which can also interchangeably be referred to as a diameter if the protrusions 66 are circular) can also vary from one configuration of the patterning layer 24 to another. For instance, in Fig 10A, the protrusions 66 havesubstantially the same width, but the width between adjacent ones of the protrusions 66 is larger in Fig 10B than in Fig 10A, thereby defining a larger of pattern-creating material receiving recess 64. In some implementations, the protrusions 66 can be referred to as ridges and the space between adjacent ones of the protrusions 66 can be referred to as furrows. In some implementations, the pattern-creating material receiving recess 64 can have a furrow width within the micrometre range, and the protrusions can have a protrusion width within the micrometre range. In some implementations, the patterncreating material receiving recess can have a width within the centimeter range, while the protrusions can have a protrusion width within the centimeter range. In some implementations, the pattern-creating material receiving recess can have a width within the millimeter range, while the protrusions can have a protrusion width within the centimeter range, or vice versa. These measurements can also be applicable to other geometrical shapes of the protrusions and pattern-creating material receiving recess. In some implementations, the configuration of the pattern-creating material receiving recess 64 and the protrusions 66 of can be selected for the culture of cardiomyocytes to enable an aligned growth of the cardiomyocytes. In some implementations, the configuration of the pattern-creating material receiving recess 64 and the protrusions 66 of can be selected for the culture of muscle cells. The configuration of the pattern-creating material receiving recess 64 and the protrusions 66 can thus be adjusted for the desired application of the three-dimensional patterns that will be obtained with the patterning layer 24. In some implementations, the expression “aligned growth” is considered to encompass implementations in which the combination of the pattern-creating material receiving recess and the protrusions form a honeycomb structure or alignment, and implementations where the protrusions enable cell growth therebetween.

[0196] The patterning layer 24 can be made of various materials. When the longitudinally-extending through-hole 26 is present in the elongated body 12 and used to receive therein a patterning layer-creating material, the patterning layer-creating material can be a patterning layer-creating material that can be provided in a pre-cast form and that can subsequently transition to a cast form following incubation for a casting time period. In other words, the patterning layer-creating material can be chosen so that it can be introduced, or poured, into the longitudinally-extending through-hole 26 and reach the patterning layer cavity 20 and the patterning mold 36, and then, after the casting time period, the patterning layer-creating material transitions to a cast form to produce apatterning layer that can maintain its shape once the patterning mold 36 is removed. The patterning layer-creating material can thus initially be in a substantially liquid form, and then harden following the casting time period and under curing conditions. In some implementations, the patterning layer-creating material can include a polymeric material, which can be an elastomeric material. In some implementations, the elastomeric material can include polydimethylsiloxane (PDMS). In some implementations, the patterning layercreating material can be for instance a polymerizing solution, any soft polymer such as silicone-based polymers, rubber materials, biological materials including for instance gelatine, agarose, collagen, etc. Thus, any material that can be moldable against the patterning mold top surface 38 of the patterning mold 36 to create the pattern-creating material receiving recess 64 and the pattern-creating material receiving recess of the patterning layer 24 can be suitable for use as the patterning layer-creating material.

[0197] In other implementations, for instance when the longitudinally-extending through- hole 26 is not used to form the patterning layer 24 in situ within the patterning layer cavity 20 or when the longitudinally-extending through-hole 26 is not present, the patterning layer-creating material can be any type of material that can provide a suitable interaction between the pattern-creating material receiving recess and protrusions and the patterncreating material, such as a material that can enable retaining the pattern-creating material within the pattern-creating material receiving recess. Examples of patterning layercreating materials can include metallic materials and polymeric materials, for instance.

[0198] In some implementations, the patterning layer 24 can be a removable patterning layer. The patterning layer holder 10 can thus be used repeatedly with different configurations of patterning layers depending on the needs of the user. Thus, once the user has created the desired number of three-dimensional patterns in cell culture wells and a change in the configuration of the patterning layer is required, the user can simply remove the patterning layer 24 from the patterning layer cavity 20, for instance using the lateral openings 40 defined in the peripheral wall 18 of the patterning layer engaging portion 14, and prepare a new patterning layer using the longitudinally-extending through- hole 26 or using another pre-made patterning layer. The removable patterning layer can thus be used for a desired number of times and then be changed for another patterning layer to have an opportunity to use another predetermined spatial distribution of pattern-creating material receiving recess and protrusions specific to the three-dimensional pattern that would now be desired.

[0199] In some implementations, the patterning layer 24 can be used in an upward orientation, while in other implementations, the patterning layer 24 can be used in a downward orientation. For instance, in Figs 1 and 25, the combination of the patterning layer holder 10 and the patterning layer 24 is shown in an upward orientation, with the distal portion 62 of the patterning layer 24 being oriented to contact a pattern-creating material received in a cell culture well, for instance. In Figs 2 and 31 , the combination of the patterning layer holder 10 and the patterning layer 24 is shown in a downward orientation, with the distal portion 62 of the patterning layer 24 being oriented so as to enable receiving a pattern-creating material thereon. In such implementations, the distal portion 62 of the patterning layer 24 can be configured with a containment feature enabling retaining the pattern-creating material within the pattern-creating material receiving recess 64 of the patterning layer 24, such as a peripheral wall having a given height and width. Such peripheral wall can simply be one of the protrusions 66 that runs along the outer periphery of the patterning layer 24.

[0200] In some implementations, for instance when the patterning layer is used in an upward orientation, the casting of the pattern-creating material within the pattern-creating material receiving recess 64 of the patterning layer 24 can be done directly, with the pouring of the pattern-creating material onto the surface of the distal portion 62 of the patterning layer that includes the pattern-creating material receiving recess 64 and the protrusions 66, / .e., the pattern-creating surface.

[0201] In some implementations, the pattern-receiving surface 68 can be either hydrophobic or hydrophilic.Description of a system for preparing three-dimensional patterns in cell culture wells of a cell culture plate

[0202] Referring now to Figs 16 to 23, a patterning system 90 for preparing three- dimensional patterns in cell culture wells of a cell culture plate is shown. It is to be understood that although a cell culture plate is shown as an exemplary implementation, the method can also be applicable to other types of pattern-receiving surfaces.

[0203] In each illustrated implementation, a given number of patterning layer holders 10 is shown in cooperation with a common top platform 70 provided at respective proximal ends 28 of the elongated bodies 12 of the patterning layer holders 10 such that the elongated bodies 12 of the patterning layer holders 10 extend downwardly from the common top platform 70.

[0204] In Figs 16 and 17, four patterning layer holders 10 are shown engaged with a common top platform 70, the four patterning layer holders 10 being provided in a 2-by-2 configuration. In Figs 18 and 19, six patterning layer holders 10 are shown engaged with a common top platform 70, the six patterning layer holders 10 being provided as an array. In Figs 20 and 21 , twelve patterning layer holders 10 are shown engaged with a common top platform 70, the four patterning layer holders 10 being provided in a 6-by-2 configuration. In Figs 22 and 23, sixteen patterning layer holders 10 are shown engaged with a common top platform 70, the four patterning layer holders 10 being provided in a 4- by-4 configuration.

[0205] In some implementations, the number of patterning layer holders 10 engaged with the common top platform 70 can be determined in accordance with the number of cell culture wells of the cell culture plate that the patterning system is intended to be used with. In other words, for a cell culture plate having four cell culture wells, the patterning system can include four patterning layer holders 10, for a cell culture plate having twelve cell culture wells, the patterning system can include twelve patterning layer holders 10, for a cell culture plate having twenty-four cell culture wells, the patterning system can include twenty-four patterning layer holders 10, for a cell culture plate having forty-eight cell culture wells, the patterning system can include forty-eight patterning layer holders 10, for a cell culture plate having ninety-six cell culture wells, the patterning system can include ninety- six patterning layer holders 10, and so on.

[0206] In some implementations, the common top portion 70 and the patterning layer holders 10 form a monolithic structure, / .e., the common top portion 70 and the patterning layer holders 10 form a monolithic array of patterning layer holders. In some implementations, the patterning layer holder 10 can itself also be a monolithic structure. In some implementations, the patterning layer holder 10 can be made by an integral molding process or by an additive manufacturing process. In some implementations, thepatterning layer holder can be made using a mold fabricated by an additive manufacturing process. In some implementations, the monolithic array of patterning layer holders can be made by an integral molding process or by an additive manufacturing process. In some implementations, the patterning layer holder and optionally the common top pattern if forming a monolithic array of patterning layer holders can be made of a polymeric material such as polyvinyl alcohol (PVA), acrylonitrile butadiene styrene (ABS), polypropylene (PP), chlorinated polyethylene (CPE), polyacrylonitrile (PAN), polylactic acid (PLA), nylon, etc.

[0207] In some implementations, a plurality of patterning system sub-units can be used to obtain a resulting patterning system with a number of patterning layer holders 10 corresponding to the number of cell culture wells into which a three-dimensional pattern is desired to be obtained. For instance, for a cell culture plate that include sixteen cell culture wells, four patterning system sub-units such as shown in Fig 16 can be used. In such implementation, the patterning system sub-units can include a patterning layer that can be the same or different depending on the given patterning system sub-unit, which can depend for instance on the resulting three-dimensional patterns that are desired in selected wells of the cell culture plate.

[0208] The patterning layer holders 10 of the patterning system 90 are as described herein. In some implementations, the common top layer 70 enables each of the patterning layer holders 10 to be engaged with a common structure feature, / .e., the common top platform, such that the patterning layer holders 10 can be moved at the same time by manipulating the common top platform 70. Similarly to the casting hole 46 described above with respect to a single patterning layer holder 10, a casting hole 46 can be defined in the common top platform 70 for each corresponding patterning layer holder 10 such that each casting hole 46 is in fluid communication with a respective one of the longitudinally- extending through-hole 26 of the patterning layer holders 10. The combination of the casting hole 46 and the longitudinally-extending through-hole 26 can thus enable the introduction, or pouring, of a patterning layer-creating material in a pre-cast form into the casting hole 46 and then downstream into the longitudinally-extending through-hole 26 to create the patterning layer 24.

[0209] When the patterning system 90 as described above is used in conjunction with a cell culture plate to prepare a three-dimensional pattern in respective cell culture wells, the patterning layer holders 10 are distributed over the common top platform 70 such that a distance between the patterning layer engaging portion 14 of each of the patterning layer holders 10 can enable insertion of the patterning layer engaging portion 14 into a corresponding cell culture well. For instance, with reference to first and second patterning layer holders, this distance can be measured from the central axis 58 of the first patterning layer holder to the central axis 58 of the second patterning layer holder, which corresponds to a well-spacing distance measured between a first well central axis of a first cell culture well and a second well central axis of a second well central axis.

[0210] The cell culture plate that is used in conjunction with the patterning system can be any type of commercially available cell culture plate, with any number of cell culture wells. In some implementations, the cell culture plate can include an electrode provided in a least one of the cell culture wells. In some implementations, the cell culture plate can be a high-throughput electrophysiology cell culture plate that includes a microelectrode array (MEA) plate, such as described in US Patent No. US 9,329,168, which is incorporated herein in its entirety. Referring to Figs 14 and 15, an example of a high-throughput electrophysiology cell culture plate 100 that includes a MEA plate is shown. The high- throughput electrophysiology cell culture plate 100 includes a plurality of cell culture wells 152 and a MEA plate 110 comprising at least one layer that includes electrodes. IN the implementation shown, the cell culture wells 152 of the high-throughput electrophysiology cell culture plate 100 have a substantially circular cross-section, with a straight sidewall 112 on one of the sides.

[0211] Referring back to Figs 11 to 13, in some implementations, the patterning layer holder 10 can be shaped to mate with the shape of the cell culture wells 152 of the high- throughput electrophysiology cell culture plate 100. The peripheral wall 18 of the elongated body 12 of the patterning layer holder 10 can thus include a well-mating surface 80 that is located at the location of the straight sidewall 112 of the cell culture wells 152 of the high- throughput electrophysiology cell culture plate 100. The peripheral wall 18 of the elongated body 12 can thus have a shape that is complementary to the shape of the cell culture well 152 of the high-throughput electrophysiology cell culture plate 100 to enable insertion of the patterning layer engaging portion 14 of the patterning layer holder 10 into the cellculture well 152 of the high-throughput electrophysiology cell culture plate 100. In addition, the tapering of the patterning layer engaging portion 14 can be adjusted to the overall shape of the cell culture well 152 of the high-throughput electrophysiology cell culture plate 100 to facilitate insertion of the patterning layer engaging portion 14 of the patterning layer holder 10 into the cell culture well 152 of the high-throughput electrophysiology cell culture plate 100. In the implementation shown, the tapering of the patterning layer engaging portion 14 includes a curved wall 82 corresponding to a curve that can be defined in the peripheral wall of the cell culture well 152 of the high-throughput electrophysiology cell culture plate 100.

[0212] It is to be understood that the other features described above regarding the patterning layer holder of Figs 1 to 9 are also applicable to the patterning layer holder 10 of Figs 11 to 13.Methods for preparing first and second three-dimensional patterns onto first and second pattern-receiving surfaces respectively of a cell culture well

[0213] Referring to Figs 24A to 24H, 25A to 25D and 26A to 26D, a method for preparing first and second sets of three-dimensional patterns onto first and second pattern-receiving surfaces respectively of cell culture wells of first and second cell culture plates to enable an aligned growth of cultured cells will now be described in more detail.

[0214] In Step 200, a pattern-creating material in a pre-cast form is poured into the cell culture wells 304 of a first cell culture plate 300. Step 200 can optionally be performed as the first cell culture plate 300 is placed over ice, as illustrated in the exemplified implementation. As mentioned above, the pattern-creating material can be a polymerizing solution, any soft polymer such as silicone-based polymers, rubber materials, biological materials including for instance gelatine, agarose, collagen, etc. Step 200 of Fig 25A illustrates the pattern-creating material 302 in one of the cell culture wells 304 of the first cell culture plate 300.

[0215] In Step 202, a patterning system as described herein is placed over the first cell culture plate 300 and moved downwardly such that the patterning layer engaging portion of the elongated body of each of the patterning layer holders is inserted into a corresponding one of the cell culture wells 304. The patterning layer engaging portionhouses therein a proximal portion of a patterning layer, such that a distal portion of the pattering layer extends outwardly from the distal end of the elongated body. The patterning layer includes pattern-creating material receiving recess and protrusions provided according to a predetermined spatial distribution specific to the three-dimensional pattern that is desired to be obtained. In the illustrated implementation, the first cell culture plate used includes forty-eight cell cultures wells 304, and the patterning system includes a corresponding number of patterning layer holders, / .e., forty-eight patterning layer holders. Step 202 of Fig 25B illustrates the pattern-creating material 302 in one of the cell culture wells 304 of the first cell culture plate 300, with the distal portion of the patterning layer being in proximity of the pattern-creating material 302. Although not shown, the distal portion of the patterning layer can be in contact with the pattern-creating material 302 at this step. This contact between the distal portion of the patterning layer and the patterncreating material 302 enables the pattern-creating material to fill in the pattern-creating material receiving recess defined in the distal portion of the patterning layer.

[0216] In Step 204, the pattern-creating material is incubated for a casting time period sufficient to transition the pattern-creating material from the pre-cast form to a cast form. Optionally, a downward pressure can be applied onto the common top platform of the patterning system to facilitate the insertion of the patterning layer engaging portion into the cell culture well and the contact between the patterning layer and the pattern-creating material 302. In some implementations, the downward pressure can be applied by placing a weight 310 onto the common platform of the patterning system. In Step 204, the patterncreating material that was initially in the pre-cast form and thus in a substantially liquid form is allowed sufficient time to polymerize and mold around the protrusions and within the pattern-creating material receiving recess of the patterning layer and to retain its shape once the patterning layer is retrieved from the cast pattern-creating material. Fig 25C illustrates the pattern-creating material surrounding the protrusions and being received within the pattern-creating material receiving recess of the patterning layer. In some implementations, the casting time period can be within the minute range. In some implementations, the casting time period can be determined in accordance with the type of pattern-creating material used and / or the properties of the pattern-creating material used. For instance, in some implementations, the casting time period can from about 5 minutes to about 90 minutes.

[0217] In Step 206, the patterning system is moved upwardly such that the patterning layer holders and associated patterning layers are retrieved from the respective cell culture wells. During Step 206 and as shown in Fig 25D, the pattern-creating material remains within the pattern-creating material receiving recess of the patterning layer (this portion of the pattern-creating material is designed by reference number 308 in Figs 25 and 26), such that a first set of three-dimensional patterns can be obtained on the respective first pattern-receiving surfaces 68 of the cell culture wells of the first cell culture plate. This first set of three-dimensional patterns is thus obtained by the retrieval of pattern-creating material in a cast form from the pattern-creating material that was initially present on the pattern-receiving surface 68 of the cell culture well, because a portion of the patterncreating material in a cast form remains within the pattern-creating material receiving recess of the patterning layer when the patterning layer is retrieved from the cell culture well. Step 206 can thus be referred to a pattern-creating material retrieval step. Optionally, in Step 206, a rinsing medium can be introduced into the cell culture wells to rinse the first set of three-dimensional patterns. Accordingly, following Steps 200 to 206 as described above, a first cell culture plate that includes a first set of three-dimensional patterns on the first pattern-receiving surfaces of the cell culture wells can be obtained.

[0218] Then, in Step 208, the same patterning system from Step 206 that includes the patterning layer holders and the corresponding patterning layers with the pattern-creating material received with the pattern-creating material receiving recess of the patterning layer is placed over a second cell culture plate 306 and moved downwardly such that the patterning layer engaging portion of the elongated body of each of the patterning layer holders is inserted into a corresponding one of the cell culture wells 314. In Step 208, the pattern-creating material received within the pattern-creating material receiving recess of the patterning layer is thus transferred from the cell culture wells 304 of the first cell culture plate 300 to the cell culture wells 314 of the second cell culture plate 306 as the patterncreating material received within the pattern-creating material receiving recess of the patterning layer 308 remains within the pattern-creating material receiving recess of the patterning layer, as shown in Fig 26A.

[0219] In Step 210, the pattern-creating material received within the pattern-creating material receiving recess of the patterning layer 308 is deposited onto the respective second pattern-receiving surfaces 68 of the cell culture wells 314 of the second cell cultureplate 306, as shown in Fig 26B. Step 210 can thus be referred to a pattern-creating material addition step, as it is the pattern-creating material that was initially in the patterncreating material receiving recess of the patterning layer that is transferred and deposited, i.e., added, to the respective second pattern-receiving surfaces 68 of the cell culture wells 314 of the second cell culture plate 306. Optionally, a downward pressure can be applied onto the common top platform of the patterning system to facilitate the deposition of the pattern-creating material that was initially in the pattern-creating material receiving recess of the patterning layer 308 onto the respective second pattern-receiving surfaces 68 of the cell culture wells 314 of the second cell culture plate 306. The downward pressure can be applied by placing a weight onto the common platform of the patterning system. In some implementations, the patterning layer can remain in proximity of the respective second pattern-receiving surfaces 68 for a time period ranging from about 1 second to about 10 minutes. This example is provided for exemplary purposes only, as the time period during which the patterning layer can remain in proximity of the respective second patternreceiving surfaces 68 can depend for instance on the type and / or properties of the patterncreating material used.

[0220] In Step 212, the patterning system is moved upwardly to disengage the patterning layer from the pattern-creating material from the patterning layer such that pattern-creating material that was initially in the pattern-creating material receiving recess of the patterning layer 308 remains onto the respective second pattern-receiving surfaces 68. As mentioned above, Step 212 can thus be referred to as a material addition step, given that the patterncreating material that was initially in the pattern-creating material receiving recess of the patterning layer 308 is added to the second pattern-receiving surfaces 68 of the cell culture wells 314 of the second cell culture plate 306, as shown in Fig 26C.

[0221] In optional Step 214, a rinsing medium can be introduced into the cell culture wells to rinse the second set of three-dimensional patterns, as shown in Fig 26D.

[0222] Referring now to Figs 27A to 27H, an alternative method for preparing first and second sets of three-dimensional patterns onto first and second pattern-receiving surfaces 68 respectively of cell culture wells of first and second cell culture plates to enable an aligned growth of cultured cells will be described in more detail.

[0223] In Step 400, a pattern-creating material in a pre-cast form is poured into the cell culture wells of a first cell culture plate, as shown in Fig 27A. Step 400 can optionally be performed as the first cell culture plate is placed over ice, as illustrated in the exemplified implementation. As mentioned above, the pattern-creating material can be a polymerizing solution, any soft polymer such as silicone-based polymers, rubber materials, biological materials including for instance gelatine, agarose, collagen, etc. The pattern-creating material is incubated for a casting time period sufficient to transition the pattern-creating material from the pre-cast form to a cast form. In some implementations, the casting time period can be within the minute range. In some implementations, the casting time period can be determined in accordance with the type of pattern-creating material used and / or the properties of the pattern-creating material used. For instance, in some implementations, the casting time period can from about 5 minutes to about 90 minutes.

[0224] In Step 402, once the casting time period is completed and the pattern-creating material is transitioned to a cast form, a patterning system as described herein is placed over the first cell culture plate and moved downwardly such that the patterning layer engaging portion of the elongated body of each of the patterning layer holders is inserted into a corresponding one of the cell culture wells, as shown in Fig 27B. The patterning layer engaging portion houses therein a proximal portion of a patterning layer, such that a distal portion of the pattering layer extends outwardly from the distal end of the elongated body. The patterning layer includes pattern-creating material receiving recess and protrusions provided according to a predetermined spatial distribution specific to the three- dimensional pattern that is desired to be obtained. In the illustrated implementation, the first cell culture plate used includes forty-eight cell culture wells, and the patterning system includes a corresponding number of patterning layer holders, / .e., forty-eight patterning layer holders. Step 204 of Fig 25C illustrates corresponding features, with the patterncreating material in one of the cell culture wells of the first cell culture plate and the distal portion of the patterning layer being pressed downwardly onto the pattern-creating material to fill in the pattern-creating material receiving recess defined in the distal portion of the patterning layer. Optionally, a downward pressure can be applied onto the common top platform of the patterning system to facilitate the insertion of the patterning layer engaging portion into the cell culture well and the contact between the patterning layer and the pattern-creating material. The downward pressure can be applied by placing a weight onto the common platform of the patterning system. Step 204 of Fig 25C furtherillustrates the pattern-creating material surrounding the protrusions and being received within the pattern-creating material receiving recess of the patterning layer.

[0225] In Step 406, the patterning system is moved upwardly such that the patterning layer holders and associated patterning layers are retrieved from the respective cell culture wells, as shown in Fig 24D. During Step 406 and as shown in Step 206 of Fig 25D, the pattern-creating material remains within the pattern-creating material receiving recess of the patterning layer (this portion of the pattern-creating material is designed by reference number 308 in Figs 25 and 26), such that a first set of three-dimensional patterns can be obtained on the respective first pattern-receiving surfaces 68 of the cell culture wells of the first cell culture plate. This first set of three-dimensional patterns is thus obtained by the retrieval of pattern-creating material in a cast form from the pattern-creating material that was initially present on the pattern-receiving surface 68 of the cell culture well, because a portion of the pattern-creating material in a cast form remains within the patterncreating material receiving recess of the patterning layer when the patterning layer is retrieved from the cell culture well. Step 406 can thus be referred to a pattern-creating material retrieval step. Optionally, in Step 406, a rinsing medium can be introduced into the cell culture wells to rinse the first set of three-dimensional patterns. Accordingly, following Steps 400 to 406 as described above, a first cell culture plate that includes a first set of three-dimensional patterns on the first pattern-receiving surfaces of the cell culture wells can be obtained.

[0226] Then, in Step 408, the same patterning system from Step 406 that includes the patterning layer holders and the corresponding patterning layers with the pattern-creating material received with the pattern-creating material receiving recess of the patterning layer is placed over a second cell culture plate and moved downwardly such that the patterning layer engaging portion of the elongated body of each of the patterning layer holders is inserted into a corresponding one of the cell culture wells, as shown in Fig 27E. In Step 408, the pattern-creating material received within the pattern-creating material receiving recess of the patterning layer is thus transferred from the cell culture wells of the first cell culture plate to the cell culture wells of the second cell culture plate as the pattern-creating material received within the pattern-creating material receiving recess of the patterning layer remains within the pattern-creating material receiving recess of the patterning layer.

[0227] In Step 410, the pattern-creating material received within the pattern-creating material receiving recess of the patterning layer is deposited onto the respective second pattern-receiving surfaces 68 of the cell culture wells of the second cell culture plate, as shown in Fig 27F. Step 410 can thus be referred to a pattern-creating material addition step, as it is the pattern-creating material that was initially in the pattern-creating material receiving recess of the patterning layer that is transferred and deposited, i.e., added, to the respective second pattern-receiving surfaces 68 of the cell culture wells of the second cell culture plate. Optionally, a downward pressure can be applied onto the common top platform of the patterning system to facilitate the deposition of the pattern-creating material that was initially in the pattern-creating material receiving recess of the patterning layer onto the respective second pattern-receiving surfaces of the cell culture wells of the second cell culture plate. The downward pressure can be applied by placing a weight onto the common platform of the patterning system. In some implementations, the patterning layer can remain in proximity of the respective second pattern-receiving surfaces 68 for a time period ranging from about 1 second to about 10 minutes. This example is provided for exemplary purposes only, as the time period during which the patterning layer can remain in proximity of the respective second pattern-receiving surfaces 68 can depend for instance on the type and / or properties of the pattern-creating material used.

[0228] In Step 412, the patterning system is moved upwardly to disengage the patterning layer from the pattern-creating material from the patterning layer such that pattern-creating material that was initially in the pattern-creating material receiving recess of the patterning layer remains onto the respective second pattern-receiving surfaces 68, as shown in Fig 27G. As mentioned above, Step 412 can thus be referred to as a material addition step, given that the pattern-creating material that was initially in the pattern-creating material receiving recess of the patterning layer is added to the second pattern-receiving surfaces 68 of the cell culture wells of the second cell culture plate.

[0229] In optional Step 414, a rinsing medium can be introduced into the cell culture wells to rinse the second set of three-dimensional patterns, as shown in Fig 27H.

[0230] Thus, in accordance with each method described in Steps 200 to 212 or Steps 400 to 412 above, it is possible to prepare first and second sets of three-dimensional patterns having substantially the same configuration (for instance, in the implementationillustrated in Figs 25 and 26, each of the three-dimensional patterns obtained includes five lines and four gaps) using a single molding step or a single embossing step of the patterncreating material around the protrusions and within the pattern-creating material receiving recess of the patterning layer, due to the succession of the material retrieval step and then the material addition step described above. Furthermore, given that the patterning layer holders are engaged via the common top platform, each manipulation of the common top platform has a corresponding effect on each of the patterning layer holders, such that a high number of first and second sets of three-dimensional patterns can be prepared simultaneously.

[0231] Each method described in Steps 200 to 212 or Steps 400 to 412 has been exemplified with a cell culture plate that includes forty-eight cell culture wells and thus a patterning system that includes a corresponding number of patterning layer holders, / .e., forty-eight patterning layer holders. It is to be understood that each method described in Steps 200 to 212 or Steps 400 to 412 can be applied to a single patterning layer holder such as shown in Figs 1 to 13. The methods can thus be applicable for any number of patterning layer holders, with each patterning layer holder producing first and second three-dimensional patterns in first and second cell culture wells respectively.

[0232] Accordingly, in some implementations, a first three-dimensional pattern can be referred to as a first set of three-dimensional patterns that includes from one to any number of three-dimensional patterns corresponding to the number of cell culture wells with which the patterning system is to be used, and a second three-dimensional pattern can be referred to as a second set of three-dimensional patterns that includes from one to any number of three-dimensional patterns corresponding to the number of cell culture wells with which the patterning system is to be used.

[0233] In some implementations, a method for preparing a three-dimensional pattern in a cell culture well of a cell culture plate can include introducing, or pouring, a patterning layer-creating material into a longitudinally-extending through-hole extending of an elongated body of a patterning layer holder to create a patterning layer received within a patterning layer cavity of a patterning layer engaging portion of the elongated body. The patterning layer includes pattern-creating material receiving recess and protrusions provided according to a predetermined spatial distribution that is specific to the three-dimensional pattern that is desired to be obtained. This step thus enables obtaining a patterning layer directly within the patterning layer cavity of the patterning layer holder.

[0234] A pattern-creating material is then poured into the cell culture well, and a distal portion of the patterning layer is inserted into the cell culture well to contact the patterncreating material and also be inserted into the pattern-creating material such that the pattern-creating material is received within the pattern-creating material receiving recess of the patterning layer.

[0235] The pattern-creating material is incubated for a casting time period sufficient to transition the pattern- creating material from the pre-cast form to a cast form, and the patterning layer combined with the pattern-creating material received within the patterncreating material receiving recess is subsequently retrieved from the cell culture well, thereby creating the three-dimensional pattern on a pattern-receiving surface of the cell culture well.

[0236] Optionally, if an additional cell culture plate with an additional three-dimensional pattern in an additional cell culture well is desired, the patterning layer combined with the pattern-creating material received within the pattern-creating material receiving recess can be inserted into the additional cell culture well, and the pattern-creating material received within the pattern-creating material receiving recess can be deposited onto the additional pattern-receiving surface of the additional cell culture well, thereby creating the additional three-dimensional pattern on the additional pattern-receiving surface.

[0237] Depending on the number of patterning layer holders used to perform the abovedescribed steps, an equivalent number of three-dimensional patterns in respective cell culture wells can be obtained, / .e., a first set of three-dimensional patterns can be obtained for a first cell culture plate, and a second set of three-dimensional patterns can be obtained for a second cell culture plate. These steps can thus advantageously enable producing the patterning layers and then preparing a first cell culture plate with the first set of three- dimensional patterns without having to successively prepare a three-dimensional pattern in each of the cell culture wells. In other words, with the same patterning system, a user can both produce the patterning layers and prepare a high-throughput first cell culture plate with a first set of three-dimensional patterns, the first set of three-dimensional patterns being prepared in a single step. In addition, a user can also prepare a secondhigh-throughput second cell culture plate with a second set of three-dimensional patterns by transferring the pattern-creating material received within the pattern-creating material receiving recess of the patterning layers into the cell culture wells of the second cell culture plate and depositing the pattern-creating material received within the pattern-creating material receiving recess of the patterning layers onto the pattern-receiving surfaces of the cell culture wells of the second cell culture plate, the second set of three-dimensional patterns also being prepared in a single step.Methods for preparing a three-dimensional pattern using a patterning layer

[0238] Referring to Figs 28 to 35, a method for preparing a three-dimensional pattern using a patterning layer will now be described in more detail. It is to be noted that the steps described below do not have to be all performed, and do not have to be performed successively. A selection of steps among the ones described can be sufficient depending on the intended application of the resulting three-dimensional pattern.

[0239] In Step 500, a pattern-creating material in a pre-cast form is shown being placed in contact with the pattern-creating surface of a patterning layer 24 as described herein. This aspect is also illustrated in Step 206 of Fig 206. In the illustrated example, the patterncreating material includes cells to be cultured. It is to be understood that in other implementations, the pattern-creating material may not include cells. The pattern-creating material can be a polymerizing solution, any soft polymer such as silicone-based polymers, rubber materials, biological materials including for instance gelatine, agarose, collagen, etc., a hydrogel, a gel solution comprising Matrigel®, Geltrex®, fibrin, collagen, or other types of basement membrane matrix or extracellular membrane matrix (ECM). The type of cells that can be included in the pattern-creating material can be any type of cells desired, in accordance with the intended use of the three-dimensional pattern of a cell culture. In some implementations, the cells can include for instance smooth muscle cells and / or skeletal muscle cells, osteoclasts, fibroblasts, myoblasts, neurons, etc. In some implementations, the cells can be provided as suspended cells in the patterncreating material.

[0240] In some implementations, contacting the pattern-creating material in the pre-cast form with the distal portion of the patterning layer can include pouring the pattern-creating material in the pre-cast form onto the distal portion of the patterning layer from above whilethe protruding features extend upwardly (as shown for instance in Fig 28). In alternative implementations, contacting the pattern-creating material in the pre-cast form with the distal portion of the patterning layer comprises inserting the distal portion of the patterning layer into a reservoir (which can be any type of receptacle such as a cell culture dish, a cell culture well, etc.) containing the pattern-creating material in a pre-cast form while the protruding features extend downwardly (as shown for instance in Fig 25).

[0241] In some implementations, when the pattern-creating material includes cells, the protrusions can provide a physical support for interacting with the cells. For instance, in some implementations, the cells can include muscle cells, and the protrusions can include a first protrusion and a second protrusion to enable the muscle cells to grow therebetween to create a self-assembly of muscle cells forming a bundle.

[0242] In Steps 502 and 504, the pattern-creating material in the pre-cast form is in contact with the pattern-creating surface of the patterning layer 24, and the patterncreating material is incubated for a casting time period sufficient to transition the pattern creating material from the pre-cast form to a cast form. Although not shown in Figs 29 and 30, and as mentioned above, the distal portion of the patterning layer 24 can include a containment feature enabling retaining the pattern-creating material within the patterncreating material receiving recess of the patterning layer 24, such as a peripheral wall having a given height and width, when the protrusions extend upwardly. Such peripheral wall can be one of the protrusions that runs along the outer periphery of the patterning layer 24, for instance. In some implementations, when the protrusions extend downwardly for instance such as shown in Figs 33 and 35, the distal portion of the patterning layer can be surface-treated (for instance to be hydrophilic) to retain the pattern-creating material within the pattern-creating material receiving recess. In some implementations, regardless of the orientation of the patterning layer, the pattern-creating surface can be hydrophilic or hydrophobic.

[0243] The casting time period can also enable the culture of the cells to adopt its intended configuration within the cast form of the pattern-creating material. In some implementations, for instance if the pattern-creating material includes muscle cells and the protrusions are used as a physical support, e.g., as pillars, for the cell culture, the incubation time can be selected so that the muscle cells can self-assemble into a muscletissue / fiber (also referred to as a muscle bundle) between adjacent ones of the protrusions.

[0244] In some implementations, contacting the pattern-creating material in the pre-cast form with the distal portion of the patterning layer can include inserting the distal portion of the patterning layer into a reservoir containing the pattern-creating material while the protrusions extend downwardly. In some implementations, the reservoir can be hydrophobic to enable retrieving the pattern-created material in the cast form from the reservoir.

[0245] In Step 506, the pattern-creating material in the cast form and containing the optional cell culture combined with the patterning layer 24 are shown engaged with a patterning layer holder provided in an upward orientation. It is to be understood that either one of Step 504 or Step 506 can be implemented, or Step 504 can be implemented prior to Step 506, / .e., prior to engaging the combination of the pattern-creating material in the cast form and containing the cell culture with the patterning layer 24. When Step 504 is implemented prior to Step 506, the displacement of the combination of the pattern-creating material in the cast form and containing the cell culture with the patterning layer 24 to the patterning layer holder can correspond to the step of transferring at least the patterncreating material in the cast form to a culture location, the culture location being the patterning layer holder. In other words, transferring the at least the pattern-creating material in the cast form to the culture location can include engaging at least a proximal portion of the patterning layer with the patterning layer holder. In alternative implementations, the casting time period can also occur when the pattern-creating material and the patterning layer are already engaged with the patterning layer holder. When the culture location is the patterning layer holder, the pattern-receiving surface 68 can be the patterning layer abutting wall, for instance. Thus, in some implementations, applying the at least the pattern-creating material in the cast form onto the patternreceiving surface can include applying the pattern-creating material in the cast form and the patterning layer onto the pattern-receiving surface in an upward orientation. In such implementations, an additional cell culture can be placed onto the pattern-creating material in the cast form, for instance if it is desired that the cell culture of the patterncreating material interacts with another type of cells. For example, an additional cell culture containing neurons can be added onto the pattern-creating material in the cast form sothat the muscle cells of the pattern-creating material can interact with the neurons so as to prepare an in vitro model of a neuromuscular junction (NMJ). If desired, a cover slip can be placed onto the cell cultures to cover the cell cultures and potentially enable the modification of the upward orientation to a downward orientation of the combination of the patterning layer 24.

[0246] In Step 508, the pattern-creating material in the cast form and the patterning layer are transferred to a culture location in accordance with another implementation. In Fig 32, the culture location is exemplified as a cell culture well 52. It is to be understood that any type of location that can be suitable to perform a cell culture can serve as a culture location. For instance, the culture location can be any type of pattern-receiving surface, such as a bottom of a receptacle, e.g., a cell culture dish, or a cell culture well as mentioned above. In such implementations, an additional cell culture can be placed onto the pattern-creating material in the cast form, for instance if it is desired that the cell culture of the patterncreating material interacts with another type of cells. For example, an additional cell culture containing neurons can be added onto the pattern-creating material in the cast form so that the muscle cells of the pattern-creating material can interact with the neurons so as to prepare an in vitro model of a neuromuscular junction (NMJ). If desired, a cover slip can be placed onto the cell cultures to cover the cell cultures and potentially enable the modification of the upward orientation to a downward orientation of the combination of the patterning layer 24. Thus, as shown in Fig 32, in Step 508, the combination of the patterning layer 24 and the cast pattern-creating material containing the cell culture can be placed in an upward orientation on the pattern-receiving surface 68, in this case the bottom surface of a cell culture well 52.

[0247] In Step 510, which can be implemented after Step 504 or Step 506 for instance, the combination of the patterning layer 24 and the pattern-creating material in the cast form optionally containing the cell culture is shown engaged with a patterning layer holder, the combination of the patterning layer 24, the pattern-creating material 302 in the cast form containing the cell culture and the patterning layer holder 10 being shown in a downward orientation. The combination of the patterning layer 24, the pattern-creating material in the cast form containing the cell culture and the patterning layer holder 10 can then be applied onto a pattern-receiving surface, which can be for instance a bottom surface of a receptacle, such as a cell culture well or a cell culture dish. In other words,applying the at least the pattern-creating material in the cast form onto the patternreceiving surface can include applying the pattern-creating material in the cast form and the patterning layer onto the pattern-receiving surface in an upward orientation. In some implementations, the patterning layer 24 may or may not be left in place following the application onto the pattern-receiving surface. If the patterning layer 24 is left in place, for instance within a receptacle, the patterning layer holder may or may not remain engaged with the patterning layer as desired. Furthermore, in some implementations, the patterning layer 24 may or may not be left in place. Another cell culture can already be present in the receptacle 52, for instance if it is desired that the cell culture of the pattern-creating material in the cast form interacts with another type of cells. For example, the patterncreating material in the cast form can be applied onto a pattern-receiving surface corresponding to an additional cell culture containing neurons already present in the receptacle 52, so that the muscle cells of the pattern-creating material can interact with the neurons of the additional cell culture to prepare an in vitro model of a neuromuscular junction (NMJ).

[0248] Step 512 illustrates an implementation of the pattern-creating material in the cast form containing the cell culture transferred to a culture location and applied onto a patternreceiving surface of a cell culture well 52. In this implementation, the patterning layer is subsequently removed from the pattern-creating material in the cast form. Steps 210 and 212 illustrate a similar implementation, with cells that may or may not be present in the pattern-creating material. Once again, an additional cell culture can be placed onto the cast pattern-creating material, or already be present in the receptacle, for instance if it is desired that the cell culture of the pattern-creating material interacts with another type of cells contained in the additional cell culture. For example, an additional cell culture containing neurons can be added onto the pattern-creating material in the cast form (or the pattern-creating material in the cast form can be deposited onto an additional cell culture containing neurons) so that the muscle cells of the pattern-creating material in the cast form can interact with the neurons to prepare an in vitro model of a neuromuscular junction (NMJ).

[0249] Step 514 illustrates an implementation of the pattern-creating material in the cast form containing the cell culture transferred and applied onto a pattern-receiving surface 68 of a receptacle, exemplified as a cell culture well 52. In this illustrated implementation,the patterning layer 24 remains within the receptacle 52. Another cell culture can already be present in the receptacle 52, for instance if it is desired that the cell culture of the pattern-creating material in the cast form interacts with another type of cells. For example, the pattern-creating material in the cast form can be applied onto a pattern-receiving surface corresponding to an additional cell culture containing neurons already present in the receptacle 52, so that the muscle cells of the pattern-creating material can interact with the neurons of the additional cell culture to prepare an in vitro model of a neuromuscular junction (NMJ).

[0250] In some implementations, the pattern-receiving surface can be hydrophilic or hydrophobic.

[0251] Several alternative implementations and examples have been described and illustrated herein. The implementations of the technology described above are intended to be exemplary only. A person of ordinary skill in the art would appreciate the features of the individual implementations, and the possible combinations and variations of the components. A person of ordinary skill in the art would further appreciate that any of the implementations could be provided in any combination with the other implementations disclosed herein. It is understood that the technology may be embodied in other specific forms without departing from the central characteristics thereof. The present implementations and examples, therefore, are to be considered in all respects as illustrative and not restrictive, and the technology is not to be limited to the details given herein. Accordingly, while the specific implementations have been illustrated and described, numerous modifications come to mind.

Claims

CLAIMS1. A system for preparing a three-dimensional pattern in a cell culture well to enable an aligned growth of cultured cells, the system comprising: a patterning layer holder, the patterning layer holder comprising: an elongated body, the elongated body comprising: a patterning layer engaging portion provided in a distal region of the elongated body and sized for insertion into the cell culture well; and a peripheral wall having an outer peripheral wall surface, the peripheral wall defining a patterning layer cavity within the patterning layer engaging portion, the patterning layer cavity being configured to house therein at least a proximal portion of a patterning layer, the peripheral wall further defining a longitudinally-extending through-hole extending from the patterning layer cavity to a proximal end of the elongated body, the longitudinally-extending through-hole being configured to receive therein a patterning layer-creating material in a pre-cast form to subsequently cast the patterning layer in situ within the patterning layer cavity.

2. The system of claim 1 , wherein the patterning layer engaging portion is a tapering patterning layer engaging portion that tapers inwardly to define a patterning layer engaging portion gap between a peripheral wall inner surface of the cell culture well and the outer peripheral wall surface that is larger than at the proximal end of the elongated body.

3. The system of claim 1 or 2, wherein the peripheral wall of the elongated body further defines a lateral opening located in the patterning layer engaging portion to provide access to the patterning layer cavity.

4. The system of any one of claims 1 to 3, wherein the elongated body is a cylindrical elongated body.

5. The system of any one of claim 1 to 4, wherein the patterning layer holder further comprises a top platform provided at the proximal end of the elongated body, thetop platform defining a casting hole in fluid communication with the longitudinally- extending through-hole of the patterning layer holder.

6. The system of claim 5, wherein the top platform comprises a cell culture well abutting wall configured to abut a top rim of the cell culture well.

7. The system of claim 5 or 6, wherein the top platform is a circular top platform.

8. The system of claim 5 or 6, wherein the top platform is a polygonal top platform.

9. The system of claim 8, wherein the polygonal top platform is shaped as a rectangular prism, a pentagonal prism, a hexagonal prism, a heptagonal prism, or an octagonal prism.

10. The system of any one of claims 5 to 9, wherein the top platform comprises at least one manipulation instrument receiving cavity defined in a top surface of the top platform to enable manipulation of the patterning layer holder by a manipulation instrument.

11. The system of claim 10, wherein the at least one manipulation instrument receiving cavity comprises a pair of manipulation instrument receiving cavities.

12. The system of claim 10 or 11 , wherein the at least one manipulation instrument receiving cavity is D-shaped.

13. The system of any one of claims 5 to 12, wherein the top platform comprises in indicia indicative of a pattern type provided on a top surface of the top platform.

14. The system of any one of claims 5 to 12, wherein the top platform comprises in indicia indicative of an orientation of the patterning layer, the indicia being provided on a top surface of the top platform.

15. The system of any one of claims 1 to 14, wherein the patterning layer holder comprises first and second patterning layer holders provided adjacent to each other, the first and second patterning layer holders having a common top platform and being provided at a distance from each other that enables insertion of the patterninglayer engaging portion of each of the first and second patterning layer holders in a corresponding cell culture well.

16. The system of claim 15, wherein the distance extends from a first central axis of the first patterning layer holder to the second central axis of the second patterning layer holder and corresponds to a well-spacing distance measured between a first well central axis and a second well central axis.

17. The system of claim 15 or 16, wherein patterning layer holder comprises a plurality of patterning layer holders corresponding to a number of cell culture wells of a cell culture plate with which the plurality of patterning layer holders is intended to be used.

18. The system of claim 17, wherein the cell culture plate includes 6, 12, 24, 48 or 96 cell culture wells.

19. The system of claim 17 or 18, wherein the plurality of patterning layer holders is a monolithic array of patterning layer holders.

20. The system of any one of claims 17 to 19, further comprising an electrode provided in at least one cell culture well of the cell culture wells of the cell culture plate.

21. The system of claim 20, wherein the cell culture plate is a high-throughput electrophysiology cell culture plate that includes a microelectrode array (MEA) plate.

22. The system of any one of claims 1 to 21 , further comprising the patterning layer.

23. The system of claim 22, wherein the patterning layer comprises protrusions defining a pattern-creating material receiving recess configured to receive a pattern-creating material therein such that the pattern-creating material received within the patterncreating material receiving recess subsequently creates the three-dimensional pattern once deposited onto a pattern-receiving surface of the cell culture well.

24. The system of claim 23, wherein the patterning layer-creating material from which is made the patterning layer comprises a polymeric material.

25. The system of claim 24, wherein the polymeric material is an elastomeric material.

26. The system of claim 25, wherein the elastomeric material comprises polydimethylsiloxane.

27. The system of any one of claims 22 to 26, wherein the patterning layer is a removable patterning layer.

28. A system for preparing a three-dimensional pattern in a cell culture well to enable an aligned growth of cultured cells, the system comprising: a patterning layer holder, the patterning layer holder comprising: an elongated body, the elongated body comprising: a patterning layer engaging portion provided at a distal end of the elongated body and being sized for insertion into the cell culture well; and a peripheral wall having an outer peripheral wall surface, the peripheral wall defining a patterning layer cavity within the patterning layer engaging portion; and a patterning layer having at least a proximal portion receivable within the patterning layer cavity, the patterning layer having a distal portion comprising protrusions defining a pattern-creating material receiving recess together provided according to a predetermined spatial distribution specific to the three-dimensional pattern, the patterning layer being configured to receive a pattern-creating material within the pattern-creating material receiving recess.

29. The system of claim 28, wherein the patterning layer is configured such that the pattern-creating material remains within the pattern-creating material receiving recess once cast and following retrieval of the patterning layer from the cell culture well to create the three-dimensional pattern on a pattern-receiving surface of the cell culture well.

30. The system of claim 28 or 29, wherein the pattern-creating material received within the pattern-creating material receiving recess is transferable onto an additional pattern-receiving surface of an additional cell culture well to create an additional three-dimensional pattern.

31. The system of any one of claims 28 to 30, wherein the pattern-creating material receiving recess comprises parallel furrows.

32. The system of any one of claims 28 to 31 , wherein the protrusions are provided as staggered protrusions.

33. The system of any one of claims 28 to 32, wherein the protrusions are provided as a grid.

34. The system of any one of claims 28 to 33, wherein the peripheral wall further defines a longitudinally-extending through-hole extending from the patterning layer cavity to a proximal end of the elongated body, the longitudinally-extending through-hole being configured to receive therein a patterning layer-creating material in a pre-cast form to subsequently cast the patterning layer in situ within the patterning layer engaging portion.

35. The system of claim 34, wherein the patterning layer-creating material from which is made the patterning layer comprises a polymeric material.

36. The system of claim 35, wherein the polymeric material is an elastomeric material.

37. The system of claim 36, wherein the elastomeric material comprises polydimethylsiloxane.

38. The system of any one of claims 28 to 37, wherein the patterning layer is a removable patterning layer.

39. The system of any one of claims 28 to 38, wherein the patterning layer holder comprises first and second patterning layer holders provided adjacent to each other, the first and second patterning layer holders having a common top platform and being provided at a distance from each other that enables insertion of the patterning layer engaging portion of each of the first and second patterning layer holders in a corresponding cell culture well.

40. The system of claim 39, wherein the distance extends from a first central axis of the first patterning layer holder to the second central axis of the second patterning layerholder and corresponds to a well-spacing distance measured between a first well central axis and a second well central axis.

41. The system of claim 39 or 40, wherein patterning layer holder comprises a plurality of patterning layer holders corresponding to a number of cell culture wells of a cell culture plate with which the plurality of patterning layer holders is intended to be used.

42. The system of claim 41 , wherein the cell culture plate includes 6, 12, 24, 48, or 96 cell culture wells.

43. The system of claim 41 or 42, wherein the plurality of patterning layer holders is a monolithic array of patterning layer holders.

44. The system of any one of claims 41 to 43, further comprising an electrode provided in at least one cell culture well of the cell culture wells of the cell culture plate.

45. The system of claim 44, wherein the cell culture plate comprises the cell culture plate is a high-throughput electrophysiology cell culture plate that includes a microelectrode array (MEA) plate.

46. A system for preparing three-dimensional patterns in cell culture wells of a cell culture plate to enable an aligned growth of cultured cells, the system comprising: a plurality of patterning layer holders, each patterning layer holder of the patterning layer holders comprising: an elongated body, the elongated body comprising: a patterning layer engaging portion provided at a distal end of the elongated body and sized for insertion into the cell culture well; and a peripheral wall having an outer peripheral wall surface, the peripheral wall defining a patterning layer receiving cavity within the patterning layer engaging portion, the patterning layer receiving cavity being configured to receive therein at least a proximal portion of a patterning layer;a common top platform provided at respective proximal ends of the elongated bodies of the plurality of patterning layer holders such that the elongated bodies of the plurality of patterning layer holders extend downwardly from the common top platform.

47. The system of claim 46, wherein the peripheral wall further defines a longitudinally- extending through-hole extending from the patterning layer cavity to a proximal end of the elongated body, the longitudinally-extending through-hole being configured to receive therein a patterning layer-creating material in a pre-cast form to subsequently cast a patterning layer in situ within the patterning layer engaging portion.

48. The system of claim 47, wherein the top platform defines a plurality of casting holes in fluid communication with a corresponding longitudinally-extending through-hole of the plurality of patterning layer holders.

49. The system of any one of claim 46 to 48, wherein the patterning layer engaging portion is a tapering patterning layer engaging portion that tapers inwardly to define a patterning layer engaging portion gap between a peripheral wall inner surface of the cell culture well and the outer peripheral wall surface that is larger than at the proximal end of the elongated body.

50. The system of any one of claims 46 to 49, wherein the peripheral wall of the elongated body further defines a lateral opening located in the patterning layer engaging portion to provide access to the patterning layer cavity.

51. The system of any one of claims 46 to 50, wherein the elongated body is a cylindrical elongated body.

52. The system of any one of claims 46 to 51 , wherein the top platform comprises a cell culture well abutting wall configured to abut a top rim of the cell culture wells.

53. The system of claim 52, wherein the cell culture plate includes 6, 12, 24, 48, or 96 cell culture wells.

54. The system of claim 46 to 53, wherein the plurality of patterning layer holders and the common top platform is a monolithic array of patterning layer holders.

55. The system of any one of claims 46 to 54, further comprising an electrode provided in at least one cell culture well of the cell culture wells of the cell culture plate.

56. The system of claim 55, wherein the cell culture plate is a high-throughput electrophysiology cell culture plate that includes a microelectrode array (MEA) plate.

57. The system of any one of claims 46 to 56, further comprising the patterning layer.

58. The system of claim 57, wherein the patterning layer comprises protrusions defining a pattern-creating material receiving recess configured to receive a pattern-creating material therein such that the pattern-creating material received within the patterncreating material receiving recess subsequently creates the three-dimensional pattern once deposited onto the pattern-receiving surface of the cell culture well.

59. The system of claim 58, wherein the patterning layer-creating material from which is made the patterning layer comprises a polymeric material.

60. The system of claim 59, wherein the polymeric material is an elastomeric material.

61. The system of claim 60, wherein the elastomeric material comprises polydimethylsiloxane.

62. The system of any one of claims 46 to 61 , wherein the patterning layer is a removable patterning layer.

63. A system for applying a pattern-creating material onto a pattern-receiving surface, the system comprising: a patterning layer having a distal portion comprising protrusions provided according to a predetermined spatial distribution specific to a three-dimensional pattern and defining a pattern-creating material receiving recess configured to receive therein a pattern-creating material in a pre-cast form when the patterning layer is at a casting location;wherein the distal portion is configured to retain the pattern-creating material within the pattern-creating material receiving recess once the pattern-creating material is in a cast form and when the patterning layer is moved from the casting location to a culture location where the pattern-receiving surface is located.

64. The system of claim 63, wherein the patterning layer is configured such that the protrusions extend upwardly to receive the pattern-creating material from above.

65. The system of claim 63, wherein the patterning layer is configured such that the protrusions extend downwardly, and the distal portion is configured to be inserted into a reservoir containing the pattern-creating material.

66. The system of claim 65, wherein the reservoir is surface-treated to be hydrophobic.

67. The system of any one of claims 63 to 66, wherein the pattern-receiving surface is hydrophobic.

68. The system of any one of claims 63 to 66, wherein the pattern-receiving surface is hydrophilic.

69. The system of any one of claims 63 to 68, wherein the pattern-creating material comprises a three-dimensional network of crosslinked hydrophilic polymers.

70. The system of any one of claims 63 to 69, wherein the protrusions have a crosssection selected between a polygonal cross-section, a substantially circular crosssection and an elliptic cross-section.

71. The system of any one of claims 63 to 70, wherein the pattern-creating material is configured to receive cells therein.

72. The system of claim 71 , wherein the cells comprising one or more of muscle cells, osteoclasts, fibroblasts, myoblasts, and neurons.

73. The system of claim 71 or 72, wherein the protrusions provide a physical support for interacting with the cells.

74. The system of claim 73, wherein the cells are muscle cells and the protrusions comprise a first protrusion and a second protrusion for the muscle cells to grow in between.

75. The system of any one of claims 63 to 74, wherein the pattern-receiving surface is part of a receptacle.

76. The system of claim 75, wherein the receptacle is a cell culture dish or a cell culture well.

77. The system of claim 75 or 76, wherein the receptacle is configured to receive an additional cell culture therein.

78. The system of any one of claims 75 to 77, wherein the receptacle is configured to receive a cell culture therein and to receive the pattern-creating material once in the cast form.

79. The system of any one of claims 75 to 77, wherein the cell culture well is configured to receive a cell culture therein and to receive the patterning later and patterncreating material once in the cast form.

80. The system of claim 78 or 79, wherein the cell culture comprises neurons.

81. The system of any one of claims 63 to 80, further comprising a patterning layer holder, the patterning layer holder comprising: an elongated body, the elongated body comprising: a patterning layer engaging portion provided at a distal end of the elongated body; and a peripheral wall having an outer peripheral wall surface, the peripheral wall defining a patterning layer cavity within the patterning layer engaging portion configured to receive at least a proximal portion of the patterning layer therein.

82. A method for preparing first and second three-dimensional patterns on first and second pattern-receiving surfaces of first and second cell culture wells respectively to enable an aligned growth of cultured cells, the method comprising: introducing a pattern-creating material in a pre-cast form into the first cell culture well; inserting a distal portion of a patterning layer into the pattern-creating material, the patterning layer comprising protrusions defining a pattern-creating material receiving recess provided according to a predetermined spatial distribution specific to the first and second three-dimensional patterns; incubating the pattern-creating material for a casting time period sufficient to transition the pattern-creating material from the pre-cast form to a cast form; retrieving the patterning layer combined with the pattern-creating material received within the pattern-creating material receiving recess from the first cell culture well, thereby creating the first three-dimensional pattern on the first pattern-receiving surface; inserting the patterning layer combined with the pattern-creating material received within the pattern-creating material receiving recess into the second cell culture well; depositing the pattern-creating material received within the pattern-creating material receiving recess onto the second pattern-receiving surface of the second cell culture well, thereby creating the second three-dimensional pattern on the second pattern-receiving surface; and optionally retrieving the patterning layer from the second cell culture well.

83. A method for preparing first and second three-dimensional patterns on first and second pattern-receiving surfaces of first and second cell culture wells respectively to enable an aligned growth of cultured cells, the method comprising: introducing a pattern-creating material in a pre-cast form into the first cell culture well;incubating the pattern-creating material for a casting time period sufficient to transition the pattern-creating material from the pre-cast form to a cast form; inserting a distal portion of a patterning layer into the pattern-creating material, the patterning layer comprising protrusions defining a pattern-creating material receiving recess provided according to a predetermined spatial distribution specific to the first three-dimensional patterns; retrieving the patterning layer combined with the pattern-creating material received within the pattern-creating material receiving recess from the first cell culture well, thereby creating the first three-dimensional pattern on the first pattern-receiving surface; inserting the patterning layer combined with the pattern-creating material received within the pattern-creating material receiving recess into the second cell culture well; depositing the pattern-creating material received within the pattern-creating material receiving recess onto the second pattern-receiving surface of the second cell culture well, thereby creating the second three-dimensional pattern on the second pattern-receiving surface; and optionally retrieving the patterning layer from the second cell culture well.

84. The method of claim 82 or 83, wherein the first and second three-dimensional patterns correspond to first and second sets of three-dimensional patterns respectively.

85. The method of claim 84, wherein a number of first three-dimensional patterns in the first set of three-dimensional patterns corresponds to a number of the first cell culture wells of a first cell culture plate.

86. The method of claim 85, wherein the first cell culture plate includes 6, 12, 24, 48, or 96 cell culture wells.

87. The method of any one of claims 84 to 86, wherein a number of second three- dimensional patterns in the second set of three-dimensional patterns corresponds to a number of the second cell culture wells of a second cell culture plate.

88. The method of claim 87, wherein the first and second cell culture plated includes the same number of cell culture wells.

89. The method of any one of claims 82 to 88, further comprising providing an electrode in at least one cell culture well of the first and second cell culture wells.

90. The method of claim 87 or 88, wherein at least one of the first and second cell culture plates comprises a microelectrode array plate.

91. The method of any one of claims 82 to 90, wherein the casting time period ranges from about 5 minutes to about 90 minutes.

92. The method of any one of claims 82 to 91, wherein incubating the pattern-creating material for the casting time period comprises applying a downward pressure onto the patterning layer during the casting time period.

93. The method of any one of claims 82 to 92, further comprising rinsing the first three- dimensional pattern with a rinsing medium.

94. The method of any one of claims 82 to 93, wherein depositing the pattern-creating material received within the pattern-creating material receiving recess onto the pattern-receiving surface of the second cell culture well comprises applying a downward pressure onto the patterning layer.

95. The method of any one of claims 82 to 94, further comprising rinsing the second three-dimensional pattern with a rinsing medium.

96. A method for preparing a three-dimensional pattern in a cell culture well of a cell culture plate, the method comprising: introducing a patterning layer-creating material into a longitudinally-extending through-hole extending of an elongated body of a patterning layer holder to create a patterning layer received within a patterning layer cavity of apatterning layer engaging portion of the elongated body, the patterning layer comprising protrusions defining a pattern-creating material receiving recess provided according to a predetermined spatial distribution specific to the three- dimensional pattern; introducing a pattern-creating material into the cell culture well; inserting a distal portion of the patterning layer into the pattern-creating material such that the pattern-creating material is received within the patterncreating material receiving recess of the patterning layer; incubating the pattern-creating material for a casting time period sufficient to transition the pattern-creating material from the pre-cast form to a cast form; and retrieving the patterning layer combined with the pattern-creating material received within the pattern-creating material receiving recess from the cell culture well, thereby creating the three-dimensional pattern on a patternreceiving surface of the cell culture well.

97. The method of claim 96, further comprising: inserting the patterning layer combined with the pattern-creating material received within the pattern-creating material receiving recess into an additional cell culture well of an additional cell culture plate; and depositing the pattern-creating material received within the pattern-creating material receiving recess onto an additional pattern-receiving surface of the additional cell culture well, thereby creating an additional three-dimensional pattern on the additional pattern-receiving surface.

98. The method of claim 96 or 97, wherein the casting time period ranges from about 5 minutes to about 90 minutes.

99. The method of any one of claims 96 to 98, wherein incubating the pattern-creating material for the casting time period comprises applying a downward pressure onto the patterning layer during the casting time period.

100. The method of claim 99, wherein depositing the pattern-creating material received within the pattern-creating material receiving recess onto the additional patternreceiving surface of the additional cell culture well comprises applying a downward pressure onto the patterning layer.

101. The method of any one of claims 96 to 100, wherein the patterning layer holder comprises first and second patterning layer holders provided adjacent to each other, the first and second patterning layer holders having a common top platform and being provided at a distance from each other that enables insertion of the patterning layer engaging portion of each of the first and second patterning layer holders in a corresponding cell culture well.

102. The method of any one of claims 96 to 101 , wherein patterning layer holder comprises a plurality of patterning layer holders corresponding to a number of cell culture wells of a cell culture plate with which the plurality of patterning layer holders is intended to be used.

103. The method of claim 102, wherein the cell culture plate includes 6, 12, 24, 48, or 96 cell culture wells.

104. The method of any one of claims 96 to 103, wherein the patterning layer holders is a monolithic array of patterning layer holders.

105. The method of any one of claims 96 to 104, further comprising providing an electrode in at least one cell culture well of the cell culture wells of the cell culture plate.

106. The method of claim 105, wherein the cell culture plate comprises a microelectrode array plate.

107. A method for applying a pattern-creating material onto a pattern-receiving surface, the method comprising: contacting, at a casting location, the pattern-creating material in a pre-cast form with a distal portion of a patterning layer, the distal portion comprising protrusions provided according to a predetermined spatial distribution specific to a three- dimensional pattern and defining a pattern-creating material receiving recess;incubating the pattern-creating material for a casting time period sufficient to transition the pattern- creating material from the pre-cast form to a cast form; and transferring at least the pattern-creating material in the cast form to a culture location to apply the at least the pattern-creating material in the cast form onto the pattern-receiving surface.

108. The method of claim 107, wherein contacting the pattern-creating material in the pre-cast form with the distal portion of the patterning layer comprises pouring the pattern-creating material in the pre-cast form onto the distal portion of the patterning layer the patterning layer while the protrusions extend upwardly.

109. The method of claim 107, wherein contacting the pattern-creating material in the pre-cast form with the distal portion of the patterning layer comprises inserting the distal portion of the patterning layer into a reservoir containing the pattern-creating material while the protrusions extend downwardly.

110. The method of claim 109, wherein the reservoir is hydrophobic.

111. The method of any one of claims 107 to 110, wherein the pattern-receiving surface is hydrophobic.

112. The method of any one of claims 107 to 110, wherein the pattern-receiving surface is hydrophilic.

113. The method of any one of claims 107 to 112, wherein the pattern-creating material comprises a three-dimensional network of crosslinked hydrophilic polymers.

114. The method of any one of claims 107 to 113, wherein the protrusions have a crosssection selected between a polygonal cross-section, a substantially circular crosssection and an elliptic cross-section.

115. The method of any one of claims 107 to 114, wherein the pattern-creating material contains cells.

116. The method of claim 115, wherein the cells comprising one or more of muscle cells, osteoclasts, fibroblasts, myoblasts, and neurons.

117. The method of claim 115 or 116, wherein the protrusions provide a physical support for interacting with the cells.

118. The method of claim 117, wherein the cells comprise muscle cells and the protrusions comprise a first protrusion and a second protrusion for the muscle cells to grow in between.

119. The method of any one of claims 115 to 118, wherein applying the at least the pattern-creating material in the cast form onto the pattern-receiving surface comprises applying the pattern-creating material in the cast form and the patterning layer onto the pattern-receiving surface in an upward orientation.

120. The method of any one of claims 115 to 118, wherein applying the at least the pattern-creating material in the cast form onto the pattern-receiving surface comprises applying the pattern-creating material in the cast form and the patterning layer onto the pattern-receiving surface in a downward orientation.

121. The method of claim 119 or 120, further comprising incubating the cells received in the pattern-creating material with an additional cell culture.

122. The method of any one of claims 115 to 118, wherein the pattern-receiving surface is part of a receptacle.

123. The method of claim 122, wherein the receptacle is a cell culture dish or a cell culture well.

124. The method of claim 122 or 123, wherein the receptacle is configured to receive an additional cell culture therein.

125. The method of claim 124, further comprising incubating the pattern-creating material in the cast form with the additional cell culture received in the receptacle.

126. The method of claim 124, further comprising incubating the pattern-creating material in the cast form combined with the patterning later with the additional cell culture received in the receptacle.

127. The method of any one of claims 124 to 126, wherein the additional cell culture comprises neurons.

128. The method of any one of claims 107 to 127, wherein at least a proximal portion of the patterning is engageable with a patterning layer holder, the patterning layer holder comprising: an elongated body, the elongated body comprising: a patterning layer engaging portion provided at a distal end of the elongated body; and a peripheral wall having an outer peripheral wall surface, the peripheral wall defining a patterning layer cavity within a patterning layer engaging portion configured to receive the at least a proximal portion of the patterning layer.

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