3D STRUCTURES FOR CELL GROWTH

Abstract

The present invention relates to a method for creating a three- dimensional structure for cell growth by creating a template of a polymer, applying a hydrogel support onto the template and removing the template. The present invention further provides a device for cell growth comprising a polymer template embedded in a hydrogel.

Claims

1. A method for creating a three-dimensional structure for cell growth comprising the steps of: a) providing a polymer; b) creating a template from the polymer; c) applying a hydrogel support onto the template to embed the template in the hydrogel; d) removing the template; wherein the polymer is a polyoxazoline which is a PsecBuOx-stat-PAOx co-polymer represented by formula (I); ##STR00005## wherein each R.sub.1 is independently selected from -methyl, -ethyl, -n-propyl, cyclo-propyl and isopropyl; the sum of m and n is from 20 to 1000, preferably from 200 to 700, most preferably from 300 to 500.

2. The method according to claim 1, wherein R.sub.1 is ethyl.

3. The method according to claim 1, wherein the polyoxazoline has a number average molecular weight of from 10 to 200 kDa.

4. The method according to claim 1, wherein the template is a fibre, cylinder or film.

5. The method according to claim 4, wherein the template comprises one or more microfilaments and the three dimensional structure obtained comprises one or more microchannels.

6. The method according to claim 5, wherein the one or more microfilaments have a diameter from 0.5 to 5000 .Math.m, preferably from 1 to 1000 .Math.m, more preferably from 5 to 20 .Math.m.

7. The method according to claim 1, wherein step (c) comprises: (i) applying an aqueous hydrogel pre-polymer on the template (ii) cross-linking the hydrogel prepolymer.

8. The method according to claim 1, wherein the method further comprises a step of seeding cells on to the template before applying the hydrogel support.

9. The method according to claim 1, wherein the step of removing the template comprises placing the template in an aqueous medium and decreasing the temperature of the template to below the LCST of the polyoxazoline polymer.

10. The method according to claim 9, wherein the temperature is decreased to below 10° C.

11. The method according to claim 1, wherein step b) comprises depositing the polyoxazoline polymer by electrospinning, fused deposition modelling, thermoforming or casting.

12. The method according to claim 1, wherein the cells are selected from the group consisting of smooth muscle cells, glial cells, vascular endothelial cells, gut epithelial cells, endometrium epithelial cells, fallopian epithelial cells, fibroblasts, macrophages, glial cells, stromal cells associated with respective epithelial tissue, neural and vascular endothelial cells.

13. Use of a three-dimensional structure obtained with the method of claim 1 for growing cells in vivo or in vitro.

14. Device for culturing cells comprising a polymer template embedded in a hydrogel, wherein the polymer is a PsecBuOx-stat-PAOx co-polymer represented by formula (I); ##STR00006## wherein each R.sub.1 is independently selected from -methyl, -ethyl, -n-propyl, cyclo-propyl and isopropyl; preferably R.sub.1 is ethyl; the sum of m and n is from 20 to 1000, preferably from 200 to 700, most preferably from 300 to 500.

15. Device according to claim 14, wherein the polymer template comprises one or more microchannels having a diameter of from 0.5 to 5000 .Math.m.

Description

DRAWINGS

[0089] FIG. 1 illustrates an outline of the generic process for making the 3D structure and an example of use with cells.

[0090] FIG. 2 illustrates different layouts of cells that can be grown within a structure obtained according to the invention.

[0091] FIG. 3 illustrates a more complex layout of a structure obtained with the process of the invention.

[0092] FIG. 4 shows the melt viscosity of different PEtOx-stat-PsecBuOx variants.

[0093] FIG. 5 shows an example of neurite growth from an IPSC-derived neural cluster grown within a device. (A) a brightfield image showing the gel device with an access well containing two IPSC clusters (Scalebar: 2 mm). (B) A fluorescence image of the same region, with neurite growth visible (Scalebar: 2 mm). (C) a close up of region of axonal extension along a microchannel within the gel (Scalebar: 100 .Math.m).

[0094] FIG. 6 shows the water contact angle at different temperatures for different polymers.

[0095] FIG. 7 shows an example of Schwann cells seeded onto a microfiber polymer template made from PEtOx-stat-PsecBuOx 20/80. Also shown is the resulting hydrogel after the template has been embedded, the collagen crosslinked, and the template dissolved after cooling to 4° C. for ~1 hr. The final 3D hydrogel culture device is comprised of microchannels lined with Schwann cells.

[0096] FIG. 1 illustrates an outline of the generic process flow for 3D hydrogel production. Starting from a template (1) of microfibers of 10 .Math.m (FIG. 1A), larger sacrificial structures (2) are added to create additional features (in this case, 3 mm pillars) (FIG. 1B). The entire structure is embedded within a hydrogel (3) (FIG. 1C), the gel is crosslinked, and the template is dissolved (D). In this way, a 3D environment is created with small scale oriented microchannels to guide nerve growth, where the larger 3 mm posts form ‘access wells’ where cell bodies can be placed so that extending neurites can grow into said channels (E).

[0097] FIG. 2 illustrates possible configurations of the cells within the 3D structure. The top row shows the top view, the bottom row shows the cross-section. FIG. 2A illustrates formation of microchannels (4) in a hydrogel (5). FIG. 2B shows cells (6) embedded in the hydrogel (5). FIG. 2C shows cells (7) coated on the microchannel. FIG. 2D shows organized cell interaction between cells embedded in the hydrogel (8) and cells coated onto the microchannel (7).

[0098] FIG. 3 shows a more complex form of the structure of FIG. 1. This is a platform with vascularization and innervation of tissue with large, perfusable channels connected to smaller cross-flow channels.

EXAMPLES

[0099] To evaluate polymers for suitability in the invention, the polymers were submitted to thermally triggered dissolution. To perform this test, initially large filaments were extruded (approximately 1 mm in diameter) using the FDM method described above with a 150 .Math.m diameter nozzle, a temperature of 200° C. and 5 Bar of applied pressure. The system used to extrude was a Bioinicia LE-100 Electrospinning system with custom MEW hardware consisting of a band heater controlled with a Temptron PID controller, which heats a metal syringe that is supported above the flat collector from the XY gantry system. These were placed within a Peltier heating/cooling element capable of maintaining liquid at temperatures ranging from 50° C. to approximately 4° C. This test allows to emulate the intended process flow where cells are seeded on the template at 37° C. And then the entire device is placed in a standard refrigerator (typically at 5° C.) to trigger template dissolution.

Comparative Example PnPrOx

[0100] Polyn-propyl-2-oxazoline) with a molecular weight of 50 kg/mol was tested. This polymer has a LCST of about 30° C. Solubility was tested in a PBS solution. A filament was prepared via FDM as described above. Briefly, the polymer was heated to 200° C. within a metal syringe and extruded through a 150 .Math.m diameter brass 3D printing nozzle with 5 Bar of air pressure. The filament was exposed to 37° C. for 10 minutes and then rapidly cooled to 5° C.

[0101] While the filament is maintained at 37° C., one can observe a change in filament opacity as it slowly absorbs some water but still maintains mechanical and morphological integrity. During the cooling process, one can observe the filament becoming rapidly more translucent as the material becomes increasingly more hydrophilic and, therefore, more soluble. However, it was observed that complete dissolution was only achieved by maintaining the filament at 5° C. for approximately 3 hrs.

[0102] In order to emulate a cell seeding process, whereby the template scaffold is seeded with cells prior to embedding and dissolution, we studied if the filament could be maintained at 37° C. for an extend period.

[0103] After maintaining the filament for 1 hr at 37° C., it was found that the filament no longer dissolved. This was ascribed to be a consequence of structural reorganization, i.e. of the semi-crystalline character of this polymer, resulting in partial crystallization. By maintaining the polymer at 37° C. (close to its Tg = 40° C.), the side changes were able to reorganize the fiber surface leading to hydrophobic fibres that can no longer dissolve.

[0104] A follow up experiment used smaller MEW generated fibres (approximately 20 .Math.m) using a 150 .Math.m diameter 3D printing nozzle, a temperature of 190° C., 0.25 Bar of pressure, -4 kV of applied voltage, 5 mm working distance, and a translation speed of 75 mm/s. Observing these fibres under similar dissolution conditions found that this phenomenon was consistent and not dependent on fibre/filament size or differences in surface-to-volume ratio (data not shown). For both sizes, samples were kept in the refrigerator for 3 days and the material still did not dissolve (data not shown).

Further Comparative Examples

[0105] Further polyoxazoline variants were tested. The polymers had a number average molar mass above 30 kg/mol and were processed into fibers, using a 150 .Math.m diameter 3D printing nozzle as described above for PnPrOx. The thermoresponsive dissolution behavior of the polymers was investigated in water with the following outcomes: [0106] PcPrOx: Poly(2-c-Propyl-2-oxazoline); LCST ~30° C. [0107] Fast dissolution upon contact with water at 37° C., not suitable [0108] PEtOx-stat-PnPrOx: Poly(2-ethyl-2-oxazoline)-stat-Poly(2-n-propyl-2-oxazoline; LCST 24-60° C. [0109] Fast dissolution upon contact with water when above the LCST, not suitable. [0110] PEtOx-stat-PnBuOx: Poly(2-ethyl-2-oxazoline)-stat-Poly(2-n-butyl-2-oxazoline); LCST 20-30° C.: [0111] Fast dissolution upon contact with water above the LCST at 37° C. or 42° C., not suitable. [0112] PsecBuOx: Poly(2-sec-Butyl-2-oxazoline) LCST ~5° C. [0113] No dissolution in water at 5° C., not suitable. [0114] PEtOx-PnBuOx 70/30: Poly(2-ethyl-2-oxazoline)-stat-Poly(2-n-Butyl-2-oxazoline); LCST 22° C. [0115] Immediate dissolution upon contact with water, not suitable. [0116] 50:50 physical blend of Poly(2-ethyl-2-oxazoline) and PsecBuOx: Poly(2-sec-Butyl-2-oxazoline); LCST 5-60° C. [0117] Immediate dissolution upon contact with water, not suitable.

Example 1 Poly(2-ethyl-2-oxazoline-stat-poly(2-sec-Butyl-2-oxazoline) (PEtOx-stat-PSecBuOx 30/70)

[0118] For this example, a copolymer of PEtOx and PSecBuOx was used with a molar ratio of 30/70 with an LCST of ~20° C. A filament was made of this copolymer as described above using a 150 .Math.m diameter brass 3D printing nozzle connected to a metal syringe heated to 205° C. through which the molten polymer was extruded with 5 Bar of air pressure. Immersed in 37° C. PBS, the filament of this polymer maintained both its shape and mechanical properties. It also retained air bubbles on its surface, indicating hydrophobicity. This was maintained for 10 minutes, after which a rapid cooling phase showed the polymer beginning to change shape, losing the air bubbles on the surface, and then begin to dissolve starting at around 20° C. With the polymer maintained at 5° C., dissolution was complete after 5 minutes.

[0119] This dissolution assay shows that this polymer has the desired properties. A test of PEtOx-stat-PSecBuOx 30/70 kept at 37° C. overnight in PBS showed that the polymer still dissolved at 5° C.

[0120] Further fibres were manufactured with MEW with diameters from 15 to 20 .Math.m. With a 150 .Math.m diameter brass 3D printing nozzle, a deposition speed of 50 mm/s, a voltage of -7 kV, working distance of 10 mm, and a pressure of 1 Bar (100 kPa), temperatures were varied resulting in the following fibre diameters.

TABLE-US-00002 Temp (°C) Diameter (.Math.m) 193 14 ± 0.14 196 15 ± 0.1 200 18 ± 0.05

[0121] Further dissolution data were obtained for the polymers shown below in Table 1:

TABLE-US-00003 Polymer PEtOx/PsecBuOx Polymer Format Preincubation Temp. Preincubation Time Swelling Temp. Dissolution Temp. Dissolution Time 1 30:70 Filament 37° C. 10 min 19° C. 5° C. 18 min 2 30:70 Fibres 37° C. 10 min 29° C. 18° C. 9 min 3* 30:70 Fibres 37° C. 16 hrs n/a 4° C. 30 min 4* 20:80 Fibres 21° C. 10 min n/a 4° C. ~1 hr 5* 20:80 Fibres 37° C. 30 min n/a 4° C. ~1 hr 6* 20:80 Fibres 37° C. 2 hrs n/a 4° C. ~1 hr 7* 10:90 Fibres 37° C. 2 hrs n/a 4° C. ~1 hr *These experiments were performed in an application setting, where the incubations and temperatures were applied as it would be during template use.

Example 2 Rheology Data for PEtOx-stat-PsecBuOx 30/70;50/50;20/80

[0122] The viscosity of the polymer melt determines the flow rate for a pressure driven MEW system, were flows from 0.5 to 0.05 ml/hr are achieved by applying pressures from 0.5 to 1.5 Bar. For a more defined process parameter, melt viscosity was measured for the different PEtOx-stat-PsecBuOx variants with a parallel plate rheometer with heated plates. 100 mg of polymer was loaded between the plates, the temperature was raised to 200° C. to initially melt the polymer, and then the viscosity was determined by measuring the rotational force required to subject the polymer melt to a cyclic of 1° angular displacement. The sample was then cooled slowly by 1° C./min. This data shows that, for the same typical range of operating temperatures (from 190 to 200° C.) the PEtOx-stat-PsecBuOx 30:70 and PEtOx-stat-PsecBuOx 20:80 achieve approximately similar melt viscosity. For the PEtOx-stat-PsecBuOx 50:50, a much higher viscosity is measured for the same range, indicating that lower flow rates will be generated and that higher temperatures (210-220° C.) are required for this polymer to be processed in a similar manner. The results are shown in FIG. 4.

Example 3 Formation Microchannels and Cell Growth

[0123] In order to test the polymer of Example 1 (PEtOx-stat-PSecBuOx 30/70) embedded in a hydrogel, a polymer mould was manufactured having polycaprolactone (PCL) pillars on a frame (poly methyl methacrylate PMMA).

[0124] Aligned fibres of the polymer were produced with MEW having a diameter of 15 .Math.m.

[0125] The pillars were placed on top of aligned fibres and, taking advantage of the relatively low melt temperature of PCL (Tm=~65° C.) compared to polyoxazolines, this assembly was placed on a hotplate held between 65° and 80° C. The bottom of the pillars were therefore exposed to a temperature above the PCL Tm and began to soften and flow, merging with the underlying fibres. After 2 to 10 seconds, the assembly was removed from the hotplate and allowed to cool. The result was an array of aligned fibres suspended between PCL pillars and, with the help of the PMMA frame, this was suspended into a hydrogel mould.

[0126] An example hydrogel that has been successfully used is rat tail collagen type 1, at a final concentration of 4 mg/ml in PBS. This pre-gel solution is typically maintained on ice (~0° C.) to maintain a liquid solution. Once this solution is heated to 37° C., a self-assembly of collagen proteins is triggered and causes the solution to form a stable, irreversible gel. Crosslinking begins to notably occur around 20° C., albeit slowly.

[0127] For this application, the collagen pre-gel solution is pre-warmed to 15° C., giving a pot-life of approximately 15 minutes before collagen crosslinking becomes too viscous to be pipetted but warm enough to limit template dissolution upon hydrogel application. This solution is then applied to the template and everything is quickly warmed to 37° C. to finalize crosslinking.

[0128] In general, it’s better to apply the hydrogel pre-polymer solution above the LCST of the template polymer, when possible. When not possible, then having the temperature as close to the LCST is a workable compromise.

[0129] Once the gel was applied and crosslinked, the device was cooled to 5° C. for approximately 15 to 30 minutes to complete dissolution. Once the fibres were dissolved, the PCL was no longer bound to the gel and the PCL+PMMA frame was removed, leaving behind a hydrogel with defined access wells connected by microchannels.

[0130] Next, IPSC-derived human neural cells were placed in the resulting access well. The outgrowth of neurons through the resulting channels was recorded and is shown in FIG. 5.

Example 4 Determination of Hydrophobicity

[0131] Water contact angle was measured to determine the hydrophobicity of the polymer at different temperatures. This confirms the LCST behaviour and also allows one to estimate the ability of cells to adhere to the polymer surface, since it is widely recognized that cells prefer a moderately hydrophobic surface (40° to 60° WCA). This was measured by spin coating a thin film of the polymer (dissolved in chloroform) onto a glass coverslip and then placing this coating coverslip onto a flat Peltier element. This was incorporated into a WCA measurement system along with a heated syringe of PBS which maintained PBS solution at approximately 60° C. A drop of this warmed PBS was deposited onto the polymer film and the WCA was monitored via time lapse over a 2 minute period until the angle had stabilize. The angle was automatically according to the proprietary software of the WCA system. The WCA of the last 30 seconds was observed to be stable and, therefore, averaged to produce the ‘final’ WCA. This was measured for a number of temperatures ranging from 37° C. to 5° C., reflecting the transition from cell incubator to refrigerator, respectively.

[0132] Data for the polymer of Example 1 (PEtOx-stat-PSecBuOx 30/70) were compared with state of the art polymer poly(N-isopropylacrylamide) (pNIPAM) and a different poly-oxazoline not according to the invention: poly(2-n-propyl-2-oxazoline, (PnProOx) with a Mw of 50 kDa. The results are shown in FIG. 6.

Example 5 Cell Seed Efficacy

[0133] Further experiments were done to evaluate cell seeding efficiency. After preparing a solution of PEtOx-stat-PSecBuOx 30/70 in water, this was added to a tissue culture well plate and the water was allowed to evaporate, forming a thin film on the bottom of each well. Primary rat Schwann cells were seeded in each well and allowed to adhere and grow over a 3 day period. Cells appeared to adhere well, though the cell morphology was not comparable to normal tissue culture plastic.

[0134] After seeding and maintenance for 3 days, the well plate was cooled to 4° C. for 15 minutes to allow the polymer to dissolve. The culture medium was collected and spun down to collect the cells in the bottom of a 15 ml tube. Cells were carefully collected, resuspended in clean medium and replaced in a fresh wellplate. They were observed to adhere again, indicating that they had survived the process and remained viable.

Example 6 Polymer Synthesis

Monomer Synthesis and Purification

[0135] 2-Ethyloxazoline (EtOx; Polymer Chemistry Innovations) was purified via fractional distillation and purification over barium oxide. 2-sec-butyloxazoline (secBuOx) was synthesized via the Witte-Seeliger method (Witte et al., Ann. Chem. 1974), from their corresponding nitrile, i.e. 2-methylbutyronitrile. The purification of secBuOx was carried out similarly to that of EtOx.

Initiator

[0136] Trifluoromethanesulfonic acid was purchased from Sigma Aldrich and used as received.

Polymerization

[0137] Polymers were synthesized with a target number of repeating units typically from 300 to 500. A typical polymer synthesis involves the administration of secBuOx monomer and a comonomer, such as EtOx, in a microwave reaction vial under an inert atmosphere. Both monomers are dosed in the desired molar ratio. Subsequently, the initiator is added in the required quantity to match the desired polymer length.

[0138] The vial is sealed under an inert atmosphere and placed in a microwave reactor (Biotage Initiator) at a temperature of 120° C. for 60 minutes.

Representative Example of a Polymerization: Poly[(2-ethyl-2-oxazoline).SUB.120.-stat-(2-sec-butyl-2-oxazoline).SUB.280 (PEtOx_{120.-stat-PsecBuOx.SUB.280.)}

[0139] An oven dried 20 mL microwave reactor vial is transferred to a glovebox (Vigor technologies) with a water content below 0.1 ppm. The vial is loaded with a stirring bar, 3.060 mL of EtOx (3.005 g, 30.3 mmol) and 9.67 mL of sec-BuOx (8.99 g, 70.7 mmol). The vial is closed and transferred out of the glovebox. A 25 mL Schlenk flask is dried, fitted with a septum, connected to a Schlenk line and filled with Argon. 10 mL of dry acetonitrile are injected into the flask, followed by 0.800 mL of trifluoromethanesulfonic acid. This stock solution is homogenized, and 0.279 mL initiator (0.038 g., 0.25 mmol) are taken with a syringe. The solution is then injected into the microwave vial containing the monomer mixture.

[0140] The vial is placed in the microwave synthesizer and heated to 120° C. for 60 minutes.

Purification and Characterization

[0141] The synthesized polymers were dissolved in dichloromethane and purified by washing three times with a saturated solution of NaHCO.sub.3 and once with water.

[0142] The polymers where characterized by 1H-NMR spectroscopy and size exclusion chromatography (SEC) on an Agilent 1260-series HPLC system equipped with a 1260 online degasser, a 1260 ISO-pump, a 1260 automatic liquid sampler (ALS), a thermo-stated column compartment (TCC) at 50° C. equipped with two PLgel 5 .Math.m mixed-D columns in series, a 1260 diode array detector (DAD) and a 1260 refractive index detector (RID). The used eluent is N,N-dimethylacetamide (DMA) containing 50 mM of lithium chloride at an optimized flow rate of 0.5 mL/min. The spectra were analyzed using the Agilent ChemStation software with the GPC add on. Molar mass (Mn and Mp) and dispersity (Ð) values were calculated against polymethylmethacrylate molar mass standards from PSS.

[0143] The characterization data for the synthesized polymers is summarized in Table 2.

TABLE-US-00004 Overview size-exclusion chromatography data for the PAOx copolymers Batch M.sub.p (PMMA) M.sub.n (PMMA) Ð T.sub.CP kDa kDa - °C PEtOx.sub.80-stat- PsecBuOx.sub.320.sup.1 49,200 28,100 2.04 13 PEtOx.sub.120-stat- PsecBuOx.sub.280.sup.2 53,000 38,600 1.97 22 PEtOx.sub.200-stat- PsecBuOx.sub.200.sup.3 101,900 43,200 2.16 30 PsecButOx.sub.300 54,000 63,000 1.14 < 4 .sup.1 Also referred to as PEtOx-stat-PSecBuOx 30/70 .sup.2 Also referred to as PEtOx-stat-PSecBuOx 20/80 .sup.3 Also referred to as PEtOx-stat-PSecBuOx 50/50

Example 7 Fiber Template Cell Seeding and Cell-Laden Channel Formation

[0144] Experiments were performed to evaluate cell seeding onto a microfiber template and subsequent embedding and dissolution of this cell-laden fiber into a collagen hydrogel to form cell-lined microchannels within the gel. After preparing microfibers of PEtOx-stat-PSecBuOx 20/80 via melt elecrowriting, this was placed in a tissue culture well plate and a cell suspension of primary rat Schwann cells in cell culture medium with 10 wt% dextran was added to the well and allowed to adhere for a 2 hr period. Cells appeared to adhere well.

[0145] The cell-laden microfiber templates were embedded in a 4 mg/ml collagen pre-gel solution at 15° C., after which the resulting device was warmed to 37° C. and held at that temperature for 30 minutes to form a crosslinked collagen hydrogel. This was then cooled to 4° C. by placing the device in a fridge for approximately 1 hr, during which time the polymer template microfibers dissolved. The resulting device consistent of a hydrogel with a 3D microchannel network with cells incorporated within the microchannels. The results are shown in FIG. 7.