A method and system for creating multi-protein patterned substrate for co-culture patterning

Abstract

The present invention discloses a method and system for multi-protein patterned substrate. The method comprising stamping a first protein layer on a substrate by a micro-stamp (102). Further, the method comprises clamping the micro-stamp (102) on the stamped substrate by a clamping device (104). Furthermore, the method comprises injecting a second protein layer on the clamped substrate by a microfluidic device (106). Thereafter, the method comprises patterning a multi-protein on the injected substrate for coculture patterning by a patterning unit (108).

Specification

Description:FIELD OF INVENTION
[001] The field of invention generally relates to a patterned substrate. More specifically, it relates to a method and system for multi-protein patterned substrate for co-culture patterning.

BACKGROUND
[002] Micro engineered patterned co-cultures are used to study interactions between different cell types and are suitable for cell patterning applications. These cellular patterns are useful in drug screening applications and offer more realistic in-vitro models for comprehending cell and tissue function. Further, microfabrication and micropatterning for cellular patterning uses a set of techniques that control cell localization on a co-culture substrate.
[003] Conventional printing techniques like stamps or molds are used to transfer patterns to a surface of a substrate. Most conventional soft lithographic techniques like microcontact printing (μCP) are limited to printing single or multiple patterns by using a single protein or ink at a time.
[004] However, printing the proteins or ink patterns one by one on the surface consumes more time and which may lead to masking of the protein deposition on the surface. Masking of the protein deposition on top of another protein pattern makes the protein pattern redundant for co-culture platform.
[005] Additionally, microfluidic systems are also used to transfer patterns to the surface of the substrate, however it is only limited to laminar flow patterning. Further, this laminar flow of the protein is limited to generating geometrical patterns in the shape of the laminarly flowing streams.
[006] Thus, in light of the above discussion, it is implied that there is need for a method and system for multi-protein patterned substrate for co-culture patterning.





OBJECT OF INVENTION
[007] The principal object of this invention is to provide a method and system to create a multi-protein pattern on substrate.
[008] A further object of the invention is to deposit the multi-protein pattern for coculturing patterning.
[009] A further object of the invention is to provide the method and system to control over the localization of cells.
[0010] Another object of the invention is to provide downstream applications, bio-micro/nano devices and biomedical engineering.

BRIEF DESCRIPTION OF FIGURES
[0011] This invention is illustrated in the accompanying drawings, throughout which, like reference letters indicate corresponding parts in the various figures.
[0012] The embodiments herein will be better understood from the following description with reference to the drawings, in which:
[0013] Figure 1 depicts a system for creating a multi-protein pattern for coculture patterning, in accordance with an embodiment of the present disclosure;
[0014] Figure 2 depicts fabrication of at least one linear groove on PDMS stamp, in accordance with an embodiment of the present disclosure;
[0015] Figure 3 depicts a schematic representation of protein transfer process using the modified micro-contact printing, in accordance with an embodiment of the present disclosure;
[0016] Figure 4 depicts a clamping unit to create the multi-protein pattern, in accordance with an embodiment of the present disclosure;
[0017] Figures 5 depicts a schematic representation of the co-culture patterning process on the multi-protein patterned surface, in accordance with an embodiment of the present disclosure;
[0018] Figures 6a-6g illustrates an exemplary quantification of percentage of adhesion of different cells to the different proteins, in accordance with an embodiment of the present disclosure;
[0019] Figure 7a-7f illustrates exemplary fluorescence microscopy images of co-culture patterns of different cell combinations on different dimensions of specific multiprotein patterns, in accordance with an embodiment of the present disclosure;
[0020] Figure 8 illustrates a method for creating a multi-protein pattern for coculture patterning, in accordance with an embodiment of the present disclosure;
[0021] Figure 9a illustrates the multi-protein pattern in checkerboard pattern, in accordance with an embodiment of the present disclosure; and
[0022] Figure 9b illustrates an exemplary fluorescence microscopy images of co-culture patterns of different cell combinations on different dimensions of specific multiprotein patterns, in accordance with an embodiment of the present disclosure.

 
STATEMENT OF INVENTION
[0023] The present invention discloses a method and system for creating a multi-protein patterns on substrate for co-culture patterning.
[0024] The method comprises stamping a first protein layer on a substrate by a micro-stamp.
[0025] Further, the method comprises clamping the micro-stamp on the stamped substrate by a clamping device.
[0026] Furthermore, the method comprises injecting a second protein layer on the clamped substrate by a microfluidic device.
[0027] Thereafter, the method comprises patterning a multi-protein on the injected substrate for coculture patterning by a patterning unit.
[0028] The proposed multi-protein patterned substrate is used for cell patterning applications like tissue engineering, drug testing, cancer research, regenerative medicine, biomaterials development, toxicology testing, cell biology research, and wound healing studies.
 
DETAILED DESCRIPTION
[0029] The embodiments herein and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments that are illustrated in the accompanying drawings and/or detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.
[0030] The present invention discloses a method and system for creating a multi-protein patterned substrate for co-culture patterning.
[0031] Figure 1 depicts a system 100 for creating a multi-protein pattern for coculture patterning. The system 100 comprises a polydimethylsiloxane (PDMS) micro-stamp 102 configured to stamp at least one of a first protein layer on the surface of a substrate 310. Wherein the PDMS micro-stamp 102 is a technique that uses the PDMS, a flexible silicone polymer, to create micro-patterned surfaces based on a desired application.
[0032] In an embodiment, the desired application comprises at least one of biology, materials science, or micro-fabrication. Further, the fabrication of PDMS micro-stamp 102 comprises at least one of a molding, a PDMS casting, and the stamping.
[0033] In one embodiment, the molding comprises creating a master mold with the desired micro-pattern. The mold can be made from materials like silicon. Herein, the master mold is also referred to as the mold.
[0034] In one embodiment, the PDMS casting comprises a process of preparing the PDMS stamp. In an embodiment, the PDMS material is poured over the master mold and cured to form a solid, and a flexible stamp. Further, the PDMS material from the mold is replicated as the PDMS stamp.
[0035] In one embodiment, the stamping comprises the process of transferring protein patterns onto different surfaces of the substrate using one or more techniques.
[0036] The one or more techniques are not limited to micro-contact printing techniques. Wherein the stamp is pressed onto the substrate coated with the protein, that can adhere to the patterned areas.
[0037] In an embodiment, the system 100 comprises the clamping unit 104. The clamping unit 104 is configured to hold the PDMS micro stamp with the first protein layer on the surface of the substrate to create the multi-protein patterned substrate 108.
[0038] Further, the system 100 comprises a microfluidic device 106. The microfluidic device 106 may be used in applications comprising at least one of Lab-on-a-Chip, cell culture systems, drug screening, and the like. Further, the microfluidic device 106 injects a second protein layer onto the substrate to form the multi-protein patterned substrate 108.
[0039] In one embodiment, the microfluidic device 106 is not limited to at least one syringe, the at least one syringe may comprises at least one of injector, dispenser, dropper, pump, ejector and spray.
[0040] In one embodiment, the system 100 comprises a multi-protein patterned substrate 108. The multi-protein patterned substrate 108 includes the first protein layer and the second protein layer to form the multi-protein patterned substrate 108. Furthermore, the multi-protein patterned substrate 108 is used for the co-culture patterning 110.
[0041] Figure 2 depicts the system 200 for fabricating the at least one linear groove on the PDMS stamp, in accordance with an embodiment. The fabrication of the at least one linear groove on the PDMS stamp comprises a silicon wafer substrate 202, a SU8 204, a PDMS 206 and the PDMS stamp 208.
[0042] In one embodiment, the silicon wafer substrate 202 provides a smooth surface and high precision surface. Further, the silicon wafer substrate 202 is initially cleaned at 80oC for a duration of twenty minutes using a piranha solution. Wherein the piranha solution comprises a mixture of sulfuric acid (H2SO4) and hydrogen peroxide (H2O2) in a ratio of 10:1. The silicon wafer substrate 202 is washed with deionized water, followed by acetone and isopropyl alcohol (IPA). Furthermore, the silicon wafer substrate 202 is allowed to dry in the presence of nitrogen gas (N2) followed by baking for dehydration on a hot plate for a duration of five minutes at a temperature of 120oC. Accordingly, the silicon wafer substrate 202 is placed within a quartz glass chamber and exposed to hexamethyldisiloxane (HMDS) for a duration of five minutes in order to enhance photoresist adherence. Wherein the HMDS is a siloxane compound generally used in one or more applications. In one embodiment, the one or more applications comprises at least one of a microfabrication and a surface treatment agent.
[0043] In an embodiment, following the HMDS treatment, the SU8 204 layer is coated onto the silicon wafer substrate 202. Wherein the SU8 204 is a photoresist material, used as an epoxy-based negative photoresist with a high aspect ratio and durability in microfabrication processes. Further, the SU8 204 is immediately spin coated at a speed of 500 rpm to 1000 rpm for a duration of 5 second to 60 second onto the silicon wafer substrate 202 and subsequently the SU8 204 is soft baked at the temperature of 95oC for a duration of 2 minute, 35 second.
[0044] In one embodiment, advantageously, applying HMDS can improve the adhesion of photoresists (like SU-8) to silicon or glass substrates, reducing defects and improving pattern quality.
[0045] Furthermore, the SU8 204 coated silicon wafer substrate 202 is exposed by UV photolithography with the help of a predesigned photomask, and subsequently, the SU8 204 coated silicon wafer substrate 202 is post baked at a temperature of 65oC to 95oC for a duration of 1 minute to 1 minute 35 second.
[0046] In one embodiment, after post baking the SU8 204 coated silicon wafer substrate 202, the SU8 204 coated silicon wafer substrate 202 is placed in a SU8 developer solution for a duration of 2 minutes. The SU8 developer solution is placed inside an ultrasonicator device (not shown) to remove the unexposed areas and form the at least one linear groove 212 on the silicon wafer substrate 202.
[0047] In an embodiment, the at least one linear groove 212 on the silicon wafer substrate 202 are referred to as the master or mold, on which as many numbers of inverted replicas can be created.
[0048] Further, the mold is salinized by being exposing to fluorinated silane vapors to passivate the surface, avoiding permanent bonding between the mold and the PDMS 206. Further, the PDMS 206 is mixed with a curing agent at a ratio of 10:1 and is poured on top of the salinized mold and baked at a temperature of 65oC for a duration of 12 hours in a hot air oven. Further, the PDMS 206 is gently peeled off from the master or mold, cut into the proper size, and punched for creating inlets and outlets, and the PDMS stamp 208 is formed. In one embodiment, the top view 210 of the PDMS stamp 208 with the at least one linear groove 212 is shown in Fig. 2.
[0049] In an alternate embodiment, the master/mold for the PDMS stamp can be fabricated using a three-dimensional printer.
[0050] The system for multi-protein patterned substrate. The system comprises a micro-stamp 102 configured to stamp the first protein layer on a substrate. Furthermore, the system comprises a clamping device 104 configured to clamp the micro-stamp 102 on the stamped substrate. The system comprises a microfluidic device 106 configured to inject, a second protein layer on the clamped substrate. Briefly, this clamping system comprises a patterning unit 108 configured to pattern a multi-protein on the injected substrate for coculture patterning.
[0051] In an embodiment, the micro-stamp may be created by the at least one linear groove through soft photolithography.
[0052] In an embodiment, one or more screws are used to tighten the clamping device 104 for stamping the first protein layer on the substrate and ensure reversible bonding between the micro-stamp 102 and the substrate.
[0053] The microfluidic device 106 comprises the at least one syringe to transfer the second protein layer through non-grooved channels of the clamped micro-stamp 102 on the substrate.
[0054] In an embodiment, the coculture patterning is based on sequential seeding of at least two cell samples on the patterned substrate.
[0055] In an embodiment, the sequential seeding of at least two cell samples on the patterned substrate is based on cell and extracellular matrix (ECM) protein binding.
[0056] Further, the system is configured to implement cell and extracellular matrix (ECM)-protein-binding criterion, the criterion comprise a first seeding module is configured to provide cells that exhibit favorable adhesion to at least one protein over another. Further, a second seeding module is configured to seed a first cell with a specific adhesion to a particular protein. Furthermore, a third seeding module is configured to seed a second cell with a non-specific adhesion, wherein the first cell favours all proteins, and the second cell has adhesion to a particular protein, wherein both the first and second cells used for coculture patterning possess similar protein affinities.
[0057] Additionally, a timing module is configured to control the seeding of the first cell with an earlier onset of adhesion to the at least one protein. Thereafter, a substrate module is configured to allow the second cell to occupy the remaining protein area on the substrate.
[0058] Figure 3 depicts the protein transfer process using the modified microcontact printing, in accordance with an embodiment. The microcontact printing (µCP) technique allows to transfer patterns onto the surface of the substrate. Further, an ink pad is placed below the PDMS stamp 208. The ink pad consists of a plain PDMS 304, and an ink layer coated on the top surface of the plain PDMS 304 to form an ink pad. In one embodiment, the ink layer consists of at least a first protein layer (302/1). Further, the first protein layer (302/1) from the inkpad is transferred to the tip of the PDMS stamp 208 by a contact transfer 306 to form the PDMS micro-stamp 308. Further, the PDMS micro-stamp 308 is placed on substrate 310 to transfer the first protein layer (302/1) pattern onto the surface of the substrate 310.
[0059] Figure 4 depicts a clamping unit 104 to create the multi-protein pattern substrate, in accordance with an embodiment. The clamping unit 104 consists of a locked clamping unit (104/1), an unlocked clamping unit (104/2), one or more screws (402/1, 402/2, 402/3, 402/4) to tighten the clamping unit 104, and a tray 403 to hold the PDMS micro-stamp 308 and the substrate 310. Further, the PDMS micro-stamp 308 and the substrate 310 are placed on tray 403 of the unlocked clamping unit (104/2), and one or more screws (402/1, 402/2, 402/3, 402/4) are screwed to tighten the clamping unit 104 as the locked clamping unit (104/1).
[0060] Furthermore, the at least one syringe injects the second protein layer (302/2) to flow through the grooved channels of the clamped micro-stamp 308. Subsequently, the multi-protein patterned substrate 108 is formed by printing the patterns of the first protein layer (302/1) and flowing the second protein layer (302/2) on the substrate 310 in the locked clamping unit (104/1). Accordingly, the PDMS micro-stamp 308 and the substrate 310 are removed from the clamping unit 104, and the PDMS micro-stamp 308 is lifted off from the substrate 310. The multi-protein patterned substrate 108 is further used for coculture patterning 110.
[0061] Figure 5 depicts the coculture patterning on the multi-protein patterned substrate, in accordance with an embodiment. The coculture patterning is based on the affinity of one or more cells 508 to different protein patterns 302, and the time at which the initial adhesion starts on the different protein patterns 302. In one embodiment, the non-protein areas on the multi-protein patterned substrate 108 are backfilled with a material 506 to avoid non-specific cell attachment. In one embodiment, the material 506 comprises of at least one of a cytophobic agent, 0.2% of Pluronic F127. In one embodiment, the multi-protein pattern is transferred to the petri dish 504 by contact transfer for coculture patterning 110. Alternatively, the multi-protein patterned substrate 108 can be used for coculture patterning 110 without transferring to the petri dish 504. In one embodiment, linear protein patterns 302 of different proteins were transferred on a petri dish 504 by microcontact printing using the micro stamps. Further, the one or more cells 508 suspension is seeded on the protein strips 302 in the petri dish 504 and studied for one or more cell 508 adhesion percentage from the initial adhesion point onward on different protein strips 302. In one embodiment, one or more cells 508 comprise at least one of the first cell 508/1 and the second cell 508/2. In one embodiment, the protein strips 302 comprise of at least one of the first protein strips 302/1 and the second protein strips 302/1.
[0062] In one embodiment, the first cell 508/1 is seeded on the protein strips 302. After the initial adhesion of maximum cells has occurred on the first protein strip 302/1, the cells 508 on the protein strip 302 were washed using a phosphate buffered saline (PBS) solution to remove unattached cells. The remaining attached cells are untouched to complete their adhesion for a duration of one hour on the protein strips 302. In one embodiment, the PBS wash is a balanced saline solution used to wash cells to maintain osmotic balance between the internal and external environment of cells.
[0063] Similarly, the second cell 508/2 is seeded on the protein strip 302. After the initial adhesion of maximum cells has occurred on the second protein strip 302/2, the cells 508 on the protein strips 302 were washed using PBS solution to remove unattached cells. The remaining attached cells are untouched to complete their adhesion for a duration of one hour on the protein strips 302. Finally, the cocultured platform of one or more cells 508 is adhered to complementary protein strips 302.
[0064] In one embodiment, the sequential seeding of one or more cells 508 onto pre-defined protein patterns 302 allows a spatial control over the localization of one or more cell 508 types.
[0065] In one embodiment, the one or more cells 508 comprise at least one of: NIH3T3 (embryonic mouse fibroblast cells), L929 (mouse fibroblast cells), HeLa (human cervical carcinoma cells), HepG2 (human hepatoma cells), SiHa (human squamous cell carcinoma cells), MCF7 (human breast cancer cells) and MG63 (human osteosarcoma cells).
[0066] In one embodiment, the protein strips 302 comprise at least one of: fibronectin 50 µg/ml (FN50), fibronectin 10 µg/ml (FN10), collagen 50 µg/ml (CN50), and laminin 50 µg/ml (LN50).
[0067] Figures 6a-6g illustrate an exemplary schematic quantification of the percentage of adhesion of one or more cells 508 adhering to the one or more proteins 302, in accordance with an embodiment. In one embodiment, the quantification of percentage of one or more cells 508 adhering to one or more proteins 302 after a duration of 15 minutes, 30 minutes and 60 minutes as shown in the figures 6a-6g. The increase in shades indicates the increase in one or more cells 508 adhesion percentage.
[0068] In one embodiment, one or more cells 508 adhering to one or more proteins 302 is based on the cell and extracellular matrix (ECM) protein binding condition in a coculture patterning 110, wherein the proteins significantly influence cell behavior and cell interactions. In one embodiment, the ECM protein binding conditions are firstly that one or more cells 508 should have favorable adhesion to one or more proteins 302 than the other. For example, NIH3T3 (cell type) has favorable adhesion to fibronectin (protein type) compared to laminin (protein type), whereas HepG2 (cell type) favors laminin (protein type) considerably. Hence Laminin - Fibronectin pattern can be done for the HepG2 - NIH3T3 pattern.
[0069] Secondly, in case the first cell favors all the proteins, and the second cell has affinity to a particular protein, then the second cell having specific affinity should be seeded first, followed by the second with non-specific adhesion. For example, MCF7 (cell type) has non-specific adhesion affinity, whereas NIH3T3 (cell type) has favorable adhesion to fibronectin (protein type). Subsequently, fibronectin- Laminin pattern is created, and NIH3T3 (cell type) is seeded first for adhesion on fibronectin (protein type), followed by MCF7 (cell type) seeding and adhesion on remaining Laminin (protein type) filled areas.
[0070] Thirdly, in case both cells for coculture have similar protein affinities, the initial adhesion time to any particular protein needs to be considered. Where one cell with early onset adhesion to a protein is seeded first and adhered, followed by the other to fill the remaining second protein area. For example, SiHa (cell type) and HeLa (cell type) have similar adhesion profiles. However, SiHa (cell type) adheres to Laminin (protein type) with a good cell adhesion percentage in 15 mins itself. Hence, on a Laminin-Fibronectin 10 µg/ml pattern, SiHa (cell type) is sedded and incubated for 15 minutes of adherence time on Laminin (protein type), after which HeLa (cell type) is seeded and adheres to Fibronectin (protein type).
[0071] Figure 7a-7f illustrates an exemplary fluorescence microscopy images of protein patterns of different dimensions, in accordance with an embodiment. Figure 7a depicts fluorescence microscopy images of HeLa-L929 (cell type) coculture on Fibronectin 50 µg/ml - Collagen 50 µg/ml (protein patterns) of different dimensions; a) 130 µm; b) 110 µm; and c) 90 µm. (i, ii and iii scale bar: 100 µm; iv, v, and vi scale bar: 200 µm) respectively.
[0072] Figure 7b depicts florescence microscopy images of HeLa-NIH3T3 (cell type) coculture on Laminin 50 µg/ml-Fibronectin 50 µg/ml (protein patterns) of different dimensions; a) 130 µm; b) 110 µm; and c) 90 µm. (i, ii and iii scale bar: 100 µm; iv, v, and vi scale bar: 200 µm) respectively.
[0073] Figure 7c depicts fluroscence microscopy images of MCF7-NIH3T3 coculture on Laminin 50 µg/ml-Fibronectin 50 µg/ml protein patterns of different dimensions; a) 130 µm; b) 110 µm; and c) 90 µm. (i, ii and iii scale bar: 100 µm; iv, v, and vi scale bar respectively.
[0074] Figure 7d depicts fluroscence microscopy images of MG63-NIH3T3 coculture on collagen 50 µg/ml-fibronectin 50 µg/ml protein patterns of different dimensions; a) 130 µm; b) 110 µm; and c) 90 µm. (i, ii and iii scale bar: 100 µm; iv, v, and vi scale bar respectively.
[0075] Figure 7e depicts fluroscence microscopy images of HepG2-NIH3T3 coculture on Laminin 50 µg/ml-Fibronectin 50 µg/ml protein patterns of different dimensions; a) 130 µm; b) 110 µm; and c) 90 µm. (i, ii and iii scale bar: 100 µm; iv, v, and vi scale bar: 200 µm) respectively.
[0076] Figure 7f depicts fluroscence microscopy images of HeLa - SiHa coculture on Laminin 50 µg/ml-Fibronectin 10 µg/ml protein patterns of different dimensions; a) 130 µm; b) 110 µm; and c) 90 µm. (i, ii and iii scale bar: 100 µm; iv, v, and vi scale bar: 200 µm) respectively.
[0077] Figure 8 illustrates a method 800 for creating a multi-protein pattern for coculture patterning, in accordance with an embodiment.
[0078] The method 800 begins with stamping, by a micro-stamp 102, a first protein layer on a substrate, as depicted at step 802.
[0079] Subsequently, the method 800 discloses clamping, by a clamping device 104, the micro-stamp 102 on the stamped substrate, as depicted at step 804.
[0080] The method 800 discloses injecting, by a microfluidic device 106, a second protein layer on the clamped substrate, as depicted at step 806.
[0081] Further, the method comprises creating the micro-stamp 102 by at least one linear groove through soft photolithography.
[0082] Furthermore, the method comprises tightening the clamping device 104 using one or more screws for stamping the first protein layer on the substrate and ensuring reversible bonding between the micro-stamp 102 and the substrate.
[0083] Furthermore, the method comprises transferring the second protein layer through non-grooved channels of the clamped micro-stamp 102 on the substrate using at least one syringe in the microfluidic device 106.
[0084] Additionally, the method comprises providing the coculture patterning based on sequential seeding of at least two cell samples on the patterned substrate.
[0085] Furthermore, the method comprises providing the sequential seeding of at least two cell samples on the patterned substrate for the co-culture patterning is based on cell and extracellular matrix ECM-protein-binding.
[0086] Furthermore, the method comprises providing a criterion for cell and extracellular matrix ECM-protein-binding. The criterion comprises providing cells that exhibit favourable adhesion to at least one protein over another by a first seeding module. Further, the criterion comprises seeding a first cell with a specific adhesion to a particular protein by a second seeding module. Furthermore, the criterion comprises seeding a second cell with a non-specific adhesion by a third seeding module, wherein the first cell favours all proteins, and the second cell favours adhesion to a particular protein, wherein both of the first cell and the second cell are used for coculture patterning possess similar protein affinities. Additionally, the criterion comprises controlling the seeding of the first cell with an earlier onset of adhesion to the at least one protein by a timing module. Thereafter the criterion comprises allowing the second cell to occupy the remaining protein area on the substrate by a substrate module.
[0087] Thereafter, the method 800 discloses patterning, by a patterning unit 108, a multi-protein on the injected substrate for coculture patterning, as depicted at step 808.
[0088] In one embodiment, the multi-protein patterned substrate 108 comprises at least one of a linear strip pattern and/or a checkerboard pattern. Further, the linear strip pattern is created through soft photolithography.
[0089] In an alternate embodiment, the checkerboard pattern is created through three-dimensional printing.
[0090] In an alternate embodiment, the multi-protein pattern comprises a checkerboard pattern as shown in fig. 9a, subsequently used for coculture patterning as shown in fig. 9b.
[0091] Figure 9a depicts the multi-protein pattern in checkerboard pattern, in accordance with an embodiment. In one embodiment, the fabrication of the check board design is patterned using three-dimensional printing techniques using a Phrozen Sonic Mini 8K Resin three-dimensional Printer. In one embodiment, the master mold is created and cured in UV for one hour duration. To prevent the permanent binding between the mold and the PDMS 206, the master mold is silanized first and subsequently is exposed to fluorinated silane vapors. Further, the PDMS 206 is mixed with the curing agent in a 10:1 ratio and poured over the silanized mold, and further baked in a hot air oven at 65 °C for twelve hours duration. Subsequently, the PDMS 206 is gently peeled off from the master or mold, cut into the proper size, and punched for creating inlets and outlets, and the PDMS stamp 208 is formed. Further, using the Phrozen Sonic Mini 8K Resin three-dimensional printer the multi-protein pattern is formed in the checkerboard pattern as shown in fig. 9a.
[0092] Figure 9b illustrates an exemplary fluorescence microscopy images of co-culture patterns of different cell combinations on different dimensions of specific multiprotein patterns, in accordance with an embodiment of the present disclosure. Fig. 9b depicts fluorescence microscopy images of HeLa-NIH3T3 coculture patterning on Laminin 50 µg/ml-Fibronectin 50 µg/ml protein patterns on checkerboard pattern for the dimensions; a) 200 µm; and b) 100 µm. (i, ii and iii scale bar: 100 µm; iv, v, and vi scale bar: 200 µm) respectively.
[0093] The advantages of the current invention include precise spatial control over cell patterning, allowing for the creation of micro engineered co-cultures suitable for high-throughput applications.
[0094] An additional advantage is that the invention enables sequential seeding of two cell populations onto predefined protein patterns, facilitating controlled coculture formation.
[0095] An additional advantage is that the modified microcontact printing technique integrates microfluidics, allowing for multi-protein patterning on the substrate.
[0096] An additional advantage is that the invention supports versatility by accommodating various cell combinations and pattern dimensions.
[0097] An additional advantage is that the method can be applied on different substrates, including glass and plasma-treated PDMS, enabling flexibility in experimental setups.
[0098] An additional advantage is that the system allows for the reversible bonding of PDMS stamps and substrates, making it reusable and cost-effective.
[0099] An additional advantage is that the multi-ink pattern process enables high-resolution patterning using different ECM proteins, ensuring optimal cell adhesion.
[00100] An additional advantage is that the invention allows for simultaneous stamping and flowing of multiple inks or proteins, streamlining the coculture preparation process.
[00101] An additional advantage is that the invention can generate patterned co-cultures with high fidelity, ensuring consistent and reproducible results.
[00102] An additional advantage is that the system is compatible with real-time cell monitoring and fluorescence microscopy for detailed observation of coculture interactions.
[00103] An additional advantage is that it accommodates different cell adhesion profiles, making it customizable for a wide range of cell types.
[00104] An additional advantage is that the patterning process minimizes non-specific cell attachment by backfilling non-protein areas with cytophobic agents like Pluronic F127.
[00105] An additional advantage is that the method can be scaled for different experimental needs, from small-scale laboratory research to larger high-throughput setups.
[00106] Applications of the current invention include tissue engineering, drug testing, cancer research, regenerative medicine, biomaterials development, toxicology testing, cell biology research, and wound healing studies.
[00107] The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the scope of the embodiments as described here.

, Claims:We claim:

1. A method for creating multi-protein patterned substrate for co-culture patterning, the method comprising:
stamping, by a micro-stamp (102), a first protein layer on a substrate;
clamping, by a clamping device (104), the micro-stamp (102) on the stamped substrate;
injecting, by a microfluidic device (106), a second protein layer on the clamped substrate;
patterning, by a patterning unit (108), a multi-protein on the injected substrate for coculture patterning, based on the stamping and the injected second protein layer.

2. The method as claimed in claim 1, comprising creating the micro-stamp (102) by at least one linear groove through soft photolithography.

3. The method as claimed in claim 1, comprising the multi-protein pattern on the injected substrate comprises at least one of a linear strip pattern or a checkerboard pattern, wherein the linear strip pattern is created through soft photolithography and wherein the checkerboard pattern is created through three-dimensional printing.

4. The method as claimed in claim 1, comprising tightening the clamping device (104) using one or more screws for stamping the first protein layer on the substrate and ensuring reversible bonding between the micro-stamp (102) and the substrate.

5. The method as claimed in claim 1, comprising transferring the second protein layer through non-grooved channels of the clamped micro-stamp (102) on the substrate using at least one syringe in the microfluidic device (106)

6. The method as claimed in claim 1, comprising providing the coculture patterning based on sequential seeding of at least two cell samples on the patterned substrate, wherein, the sequential seeding of at least two cell samples on the patterned substrate for the co-culture patterning is based on cell and extracellular matrix (ECM)-protein-binding.

7. The method as claimed in claim 6, comprising providing a criterion for cell and extracellular matrix (ECM)-protein-binding comprises:
providing cells that exhibit favourable adhesion to at least one protein over another by a first seeding module;
seeding a first cell with a specific adhesion to a particular protein by a second seeding module;
seeding a second cell with a non-specific adhesion by a third seeding module, wherein the first cell favours all proteins, and the second cell favours adhesion to a particular protein, wherein both of the first cell and the second cell are used for coculture patterning possess similar protein affinities;
controlling the seeding of the first cell with an earlier onset of adhesion to the at least one protein by a timing module; and
allowing the second cell to occupy the remaining protein area on the substrate by a substrate module.

8. A system for creating multi-protein patterned substrate for co-culture patterning, the system comprising:
a micro-stamp (102) configured to stamp the first protein layer on a substrate;
a clamping device (104) configured to clamp the micro-stamp (102) on the stamped substrate;
a microfluidic device (106) configured to inject, a second protein layer on the clamped substrate;
patterning unit (108) configured to pattern a multi-protein on the injected substrate for coculture patterning, based on the stamping and the injected second protein layer.

9. The system as claimed in claim 8, wherein the micro-stamp (102) is created by at least one linear groove through soft photolithography.

10. The system as claimed in claim 8, wherein the multi-protein pattern on the injected substrate is in at least one of a linear strip pattern or a checkerboard pattern, wherein the linear strip pattern is created through soft photolithography and wherein the checkboard pattern is created through three-dimensional printing.

11. The system as claimed in claim 8, wherein one or more screws are used to tighten the clamping device (104) for stamping the first protein layer on the substrate and ensure reversible bonding between the micro-stamp (102) and the substrate.

12. The system as claimed in claim 8, wherein the microfluidic device (106) comprises at least one syringe to transfer the second protein layer through non-grooved channels of the clamped micro-stamp (102) on the substrate.

13. The system as claimed in claim 8, wherein the coculture patterning is based on sequential seeding of at least two cell samples on the patterned substrate, wherein, the sequential seeding of at least two cell samples on the patterned substrate is based on cell and extracellular matrix (ECM)-protein-binding.

14. The system as claimed in claim 13, wherein the system is configured to implement cell and extracellular matrix (ECM)-protein-binding criterion, the criterion comprising:
a first seeding module configured to provide cells that exhibit favorable adhesion to at least one protein over another;
a second seeding module configured to seed a first cell with a specific adhesion to a particular protein;
a third seeding module configured to seed a second cell with a non-specific adhesion, wherein the first cell favours all proteins and the second cell has adhesion to a particular protein;
wherein both the first and second cells used for coculture patterning possess similar protein affinities;
a timing module configured to control the seeding of the first cell with an earlier onset of adhesion to the at least one protein; and
a substrate module configured to allow the second cell to occupy the remaining protein area on the substrate.


Date: 28th October, 2024 Signature:
Name of signatory: Nishant Kewalramani
(Patent Agent)
IN/PA number: 1420