Dissolvable porous microcarriers

Abstract

The present disclosure concerns porous microcarriers and the methods for preparing the same. The microcarriers are equipped with pores throughout to house cells and allow for a protective environment therein. The microcarriers allow for cell adherence or suspension within the pores. The microcarriers are further dissolvable, allowing for the collection of the cells therein. The microcarriers are formed through generation of carbon dioxide during rapid cross-linking. A partial digest removes a formed surface, thereby exposing the pores and allow cells access to the interior of the microcarriers.

Description

DISSOLVABLE POROUS MICROCARRIERSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. §119 of U.S. Provisional Application Serial No. 63 / 603,820 filed on November 29, 2023, the content of which are relied upon and incorporated herein by reference in their entirety.FIELD

[0002] The present disclosure relates to porous microcarriers, methods of using the same, and methods of preparing porous microcarriers for high surface area cell culturing.BACKGROUND

[0003] Cell and gene therapy markets are growing rapidly with promising treatments moving into late stage clinical trials or being commercialized. One cell therapy dose can require billions of cells or trillions of viruses. Being able to provide a large quantity of cell products in a short amount of time is critical for clinical success.

[0004] Microcarriers have be used successfully in bioprocess to generate a large quantity of cells or cell products. A disadvantage associated with microcarriers is that it exposes cells to high shear stress from stirring paddles and bead-to-bead collisions during use. Therefore, weak adherent cells and sensitive cells cannot readily be cultured using traditional microcarriers. Porous microcarriers may protect sensitive cells and culturing of suspension or semi-adherent cells. However, if the cells themselves or components inside the cells are the final products, it is very difficult if not impossible to harvest or retrieve the cells from the porous microcarriers.SUMMARY

[0005] The present disclosure concerns dissolvable porous microcarriers and the methods for preparing the same.

[0006] A 1staspect of the present disclosure, either alone or in combination with any other aspect herein, relates to a method for producing porous beads for use as cellular microcarriers, the method comprising: adding, drop wise, a polymer solution comprised of a suspended polymer and a carbonate compound into a cross-linking solution comprising an acid, wherein each drop of the polymer solution reacts with the cross-linking solution to form a porous bead, the porous bead comprising an internal porosity; and treating the porous beadwith an exfoliating solution to expose the internal porosity of the porous bead at a surface of the porous bead.

[0007] A 2ndaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 1staspect, wherein the cross-linking solution is actively stirred during the adding.

[0008] A 3rdaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 1staspect, wherein the polymer solution comprises a suspended pectic polymer.

[0009] A 4thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 3rdaspect, wherein the suspended pectic polymer comprises polygalacturonic acid.

[0010] A 5thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 3rdaspect, wherein the suspended pectic polymer comprises from about 0.1 wt% to about 5 wt% of the polymer solution.

[0011] A 6thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 5thaspect, wherein the suspended pectic polymer comprises 2 wt% of the polymer solution.

[0012] A 7thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 1staspect, wherein the carbonate compound is selected from the group consisting of calcium carbonate and sodium bicarbonate.

[0013] An 8thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 1staspect, wherein the carbonate compound comprises from 2 wt% to 30 wt% of the polymer solution.

[0014] A 9thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 8thaspect, wherein the carbonate compound comprises 20 wt% of the polymer solution.

[0015] A 10thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 1staspect, wherein the polymer solution further comprises a surfactant.

[0016] An 11thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 10thaspect, wherein the surfactant is a nonionic surfactant.

[0017] A 12thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 10thaspect, wherein the surfactant is selected from the group consisting of polysorbate 80 and pluronic F-127.

[0018] A 13thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 10thaspect, wherein the surfactant comprises from 0.2 wt% to 5 wt% of the polymer solution or the cross-linking solution.

[0019] A 14thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 10thaspect, wherein the polymer solution further comprises a surfactant at a 3:4 ratio with respect to the carbonate compound.

[0020] A 15thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 1staspect, wherein the cross-linking solution comprises a surfactant.

[0021] A 16thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 15thaspect, wherein the surfactant is a nonionic surfactant.

[0022] A 17thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 15thaspect, wherein the surfactant is selected from the group consisting of polysorbate 80 and pluronic F-127.

[0023] An 18thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 15thaspect, wherein the surfactant comprises from 0.2 wt% to 5 wt% of the polymer solution or the cross-linking solution.

[0024] A 19thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 15thaspect, wherein the polymer solution further comprises a surfactant at a 3:4 ratio with respect to the carbonate compound.

[0025] A 20thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 1staspect, wherein the carbonate compound comprises calcium to cross-link the polymer solution.

[0026] A 21staspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 1staspect, wherein the carbonate compound comprises calcium carbonate at about 0.5 to about 30 wt% of the suspended polymer weight.

[0027] A 22ndaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 1staspect, wherein the cross-linking solution further comprises a calcium salt.

[0028] A 23rdaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 22ndaspect, wherein the calcium salt is calcium chloride.

[0029] A 24thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 23rdaspect, wherein the calcium chloride comprises about 0 to about 8 wt% of the cross-linking solution.

[0030] A 25thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 23rdaspect, wherein the cross-linking solution further comprises a surfactant.

[0031] A 26thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 25thaspect, wherein the surfactant is present at a ratio of 3:4 with respect to calcium in the cross-linking solution.

[0032] A 27thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 25thaspect, wherein the surfactant is a nonionic surfactant.

[0033] A 28thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 25thaspect, wherein the surfactant is selected from the group consisting of polysorbate 80 and Pluronic f-127.

[0034] A 29thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 1staspect, wherein the acid is selected from the group consisting of acetic acid and hydrochloric acid.

[0035] A 30thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 1staspect, wherein the acid comprises about 10 wt% of the cross-linking solution.

[0036] A 31staspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 1staspect, further comprising stirring the dropped polymer solution in the cross-linking solution for a period of from about 5 minutes to about 10 hours.

[0037] A 32ndaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 1staspect, wherein each drop comprises about 0.05 pL to about 5 pL.

[0038] A 33rdaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 1staspect, further comprising collecting formed microcarriers from the cross-linking solution.

[0039] A 34thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 33rdaspect, further comprising treating the microcarriers collected from the cross-linking solution with an exfoliating solution to form exfoliated microcarriers.

[0040] A 35thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 34thaspect, wherein the exfoliating solution comprises pectinase and a chelator.

[0041] A 36thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 35thaspect, wherein the chelator comprises EDTA (ethylenediaminetetraacetic acid), ethylene glycol tetraacetic acid (EGTA), citric acid, or a combination thereof..

[0042] A 37thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 35thaspect, wherein the chelator is present at about 1 mM to about 20 mM.

[0043] A 38thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 35thaspect, wherein the pectinase is present at about 10 to about 200 U / mL.

[0044] A 39thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 36thaspect, wherein the pectinase is present at 50 U / mL.

[0045] A 40thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 34thaspect, further comprising contacting the exfoliated microcarriers with a cell.

[0046] A 41staspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 40thaspect, further comprising contacting the exfoliated microcarriers with a dissolving solution comprising a chelator and pectinase.

[0047] A 42ndaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 1staspect, wherein the internal porosity is greater than or equal to 90% of a volume of the porous bead.

[0048] A 43rdaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 1staspect, wherein the porous bead has a diameter greater than or equal to 500 pm to less than or equal to 2 mm.

[0049] A 44thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 1staspect, wherein the internal porosity comprises pores, each pore comprising a diameter greater than or equal to 50 pm to less than 500 pm.

[0050] A 45thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to a cellular microcarrier produced by the method of the 1staspect,

[0051] A 46thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to a cellular microcarrier comprising porous beads, wherein each bead comprises PGA cross-linked through calcium and pores therein, wherein the pores are from about 50 pm to about 500 pm in diameter and wherein each porous bead is from about 500 pm to about 2 mm in diameter.

[0052] A 47thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the cellular microcarrier of the 46thaspect, wherein a surface of each porous bead is exfoliated.

[0053] A 48thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the cellular microcarrier of the 47thaspect, wherein the surface of each porous bead is exfoliated by contact with a solution comprising pectinase and a chelator.

[0054] A 49thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the cellular microcarrier of the 48thaspect, wherein the pores of each porous bead are exposed on the surface.

[0055] A 50thaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the cellular microcarrier of the 49thaspect, further comprising a cell.

[0056] A 51staspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the cellular microcarrier of the 50thaspect, wherein the cell is selected from the group consisting of a mammalian cell, a bacterial cell, and an insect cell.

[0057] A 52ndaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to a cellular microcarrier comprising porous beads, wherein each bead comprises PGA cross-linked through calcium and pores therein, wherein the pores are of about 50 pm to about 500 pm in diameter, wherein each porous bead is from about 500 pm to about 2 mm in diameter, and wherein a cell is adhered within a pore.

[0058] A 53rdaspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the cellular microcarrier of the 52ndaspect, wherein the cell is selected from the group consisting of a mammalian cell, a bacterial cell, and an insect cell.

[0059] Additional features and advantages of the microcarrier and methods of preparing the same described herein will be set forth in the detailed description which follows, and in part will be readily apparent to those skilled in the art from that description or recognized by practicing the embodiments described herein, including the detailed description which follows, the claims, as well as the appended drawings.

[0060] It is to be understood that both the foregoing general description and the following detailed description describe various embodiments and are intended to provide an overview or framework for understanding the nature and character of the claimed subject matter. The accompanying drawings are included to provide a further understanding of the various embodiments, and are incorporated into and constitute a part of this specification. The drawings illustrate the various embodiments described herein, and together with the description serve to explain the principles and operations of the claimed subject matter.BRIEF DESCRIPTION OF THE DRAWINGS

[0061] FIG. la shows an example of the surface structure of samples from the Examples set forth herein, taken via SEM. Top panel-surface, middle panel- cross-section, lower panel- interior.

[0062] FIG. lb shows an example of the cross-section structure of samples from the Examples set forth herein, taken via SEM.

[0063] FIG. 1c shows an example of the interior structure of samples from the Examples set forth herein, taken via SEM.

[0064] FIG. 2a shows small (~1 mm diameter, top) microcarrier.

[0065] FIG. 2b shows FIG. 2a at higher magnification.

[0066] FIG. 2c shows a comparative large (~4 mm diameter) microcarrier.

[0067] FIG. 3a shows an SEM image of the surface of a microcarrier from the Examples set forth herein.

[0068] FIG. 3b shows an SEM image of the interior of a microcarrier from the Examples set forth herein.

[0069] FIG. 4a shows SEM images of a surface of the microcarrier from the Examples set forth herein.

[0070] FIG. 4b, like FIG. 4a, shows SEM images of the surface as described herein.

[0071] FIG. 4c shows an SEM image of the interior of the microcarriers as set forth herein.

[0072] FIG. 5a shows an SEM image of the microcarriers after 15 seconds exposure of the microcarriers to an EDTA / pectinase solution

[0073] FIG. 5b shows an SEM image of the microcarriers after 45 seconds exposure of the microcarriers to an EDTA / pectinase solution.

[0074] FIG. 5c shows an SEM image of the microcarriers after 90 seconds exposure of the microcarriers to an EDTA / pectinase solution.DESCRIPTION

[0075] The present disclosure relates to porous cellular microcarriers and methods for producing the same. In some aspects, the microcarriers are formed by a rapid cross-linkingevent that simultaneous release carbon dioxide, thereby providing pores throughout the formed spherical units. A partial digestion further removes an exterior surface of the formed microcarrier, thereby allowing cells access to the pores. The microcarriers are further advantageously dissolvable, allowing for cells suspended, adhered, or contained therein to be collected or otherwise harvested from the microcarriers. In some aspects, the present disclosure concerns a cellular microcarrier of porous beads, wherein each bead includes PGA cross-linked through calcium. Each bead includes pores therein, wherein the pores are of about 50 pm to about 500 pm in diameter and wherein each porous bead is of about 500 pm to about 2 mm in diameter. In some aspects, the surface of each porous bead is exfoliated, such as by contact with a solution comprised of pectinase and a chelator. Various embodiments of methods for making porous cellular microcarriers, porous cellular microcarriers made therefrom, and methods for using the same will be described in further detail herein with specific reference to the appended drawings.

[0076] In aspects, the present disclosure concerns providing a cellular culture with a porous microcarrier and allowing cells to culture therein. The microcarriers thereby provide a protected environment for the cells. The microcarriers of the present disclosure provide a high surface area, thereby allowing for significant cell growth within and on the microcarrier. Further, the microcarriers are dissolvable, thereby allowing a user to readily harvest or retrieve the cells from the microcarrier when desired.

[0077] In aspects of the present disclosure, microcarriers are formed at a cross-section of polymerization during effervescence in a solution. In some aspects, the microcarriers are formed by cross-linking a polymer solution in an effervescent environment such that bubbles or globules of gas therein are trapped as the polymer cross-links resulting in internal pores within the microcarrier. Furthermore, while the cross-linking reaction allows for internal pores within the microcarrier, the resulting microcarrier may be further treated to expose the internal pores and / or to remove or partially remove an outer surface thereof.

[0078] The present disclosure primarily provides two methods which can be adapted to provide porous microcarriers. Both methods are demonstrated herein to integrate porosity into dissolvable microcarrier chemistry. Both methods use a polymer solution (e.g. PGA / surfactant / carbonate) dropped into an acidic cross-linking solution (optionally with calcium, if not present in the carbonate) (or with surfactant in the cross-linking solution) to generate carbon dioxide (CO2) and create pore networks. The first method primarily uses sodium bicarbonate as a foaming agent. Sodium bicarbonate dissolves completely in anaqueous solution and thus rapidly releases carbon dioxide, making successful pore networks through to the surface. Because the formulation is a stable solution, it benefits from continuous process using pumps and nozzles. In this method, calcium is introduced in the acid solution to crosslink.

[0079] The second method uses calcium carbonate as a foaming agent, with the calcium therein allowing for cross-linking once in the acidic cross-linking solution.

[0080] It is also possible to combine these methods. Both methods produce microcarrier beads with porosities of greater than 98% (by mercury intrusion porosimetry).

[0081] In aspects, the present disclosure relates to methods for preparing porous beads for use as cellular microcarriers. In aspects, the methods include adding, drop wise, a polymer solution into a cross-linking solution. In some aspects, the polymer solution is aqueous. In some aspects, the cross-linking solution is aqueous. In some aspects, the polymer solution is comprised of a suspended polymer therein and a carbonate compound. In some aspects, the cross-linking solution comprises an acid. During the drop-wise addition, each drop of the polymer solution reacts with the cross-linking solution to form a porous bead with an internal porosity. Each formed bead can then be further treated with an exfoliating solution to expose the internal porosity of the porous bead at the surface of the porous bead. In some aspects, the cross-linking solution is actively stirred and or agitated to allow for the two solutions to come into contact.

[0082] In aspects, the polymer solution for preparing the porous microcarriers includes a suspended pectic polymer from which the porous microcarriers are formed. In some aspects, the suspended pectic polymer includes polygalacturonic acid. In some aspects, the suspended pectic polymer is from about 0. 1 wt% to about 5 wt% of the polymer solution, including about 0.2 to about 4.5 wt%, about 0.5 to about 4 wt%, about 0.8 to about 3.5 wt%, about 1.0 to about 3.0 wt%, and about 1.5 to about 2.5 wt%. In further aspects, the suspended pectic polymer is about 2 wt% of the aqueous polymer solution.

[0083] In aspects, the carbonate compound of the polymer solution is selected from the group consisting of calcium carbonate and sodium bicarbonate. In some aspects, providing a carbonate and calcium in an acidic environment allows for the rapid release of carbon dioxide as the polymer cross-links in the acidic calcium aqueous solution. In some aspects, the carbonate compound is from about 2 wt% to 30 wt% of the polymer solution, including about5 to about 25 wt%, about 10 to about 25 wt%, and about 15 to about 25 wt%. In further aspects, the carbonate compound is about 20 wt% of the polymer solution.

[0084] In some aspects, the polymer solution may further include a surfactant, such as a non-ionic surfactant. In some aspects, the presence of a surfactant can prevent or slow formed gas bubbles from coalescing into larger gas bubbles. In some aspects, the presence of a surfactant can help to control the pore size within the microcarriers. In some aspects, the surfactant is selected from polysorbate 80 and pluronic F-127. In some aspects, the surfactant is of from about 0.2 wt% to about 5 wt% of the polymer solution or the cross-linking solution, including about 0.2 to about 4.5 wt%, about 0.5 to about 4 wt%, about 0.8 to about 3.5 wt%, about 1.0 to about 3.0 wt%, and about 1.5 to about 2.5 wt%.

[0085] In some aspects, the polymer solution contains a surfactant at a weight ratio with respect to the carbonate compound therein of about 1: 10 to about 10: 1, including about 1: 1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7. 1:8, 1:9, 2: 1, 2:3, 2:5, 2:7, 2:9, 3: 1, 3:2, 3:4, 3:5, 3:7, 3:8, 3: 10, 4: 1, 4:3, 4:5, 4:7, 4:9, 5: 1, 5:2, 5:3, 5:4, 5:6, 5:7, 5:8, 5:9, 6: 1, 6:5, 6:7, 7: 1, :7:2, 7:3, 7:4, 7:5, 7:6, 7:7, 7:8, 7:9, 7: 10, 8: 1, 8:3, 8:5, 8:7, 8:9, 9: 1, 9:2, 9:4, 9:5, 9:7, 9:8, 9: 10, 10:3, 10:6, 10:7, and 10:9. In some aspects, the polymer solution contains a surfactant at a weight 3:4 ratio with respect to the carbonate compound therein.

[0086] In some aspects, the cross-linking solution includes a surfactant. In some aspects, both the polymer solution and the cross-linking solution include a surfactant. In other aspects, either the polymer solution or the cross-linking solution includes a surfactant. As with the polymer solution, the surfactant of the cross-linking solution may be a non-ionic surfactant. In some aspects, the surfactant is selected from a polysorbate, such as polysorbate 20 or polysorbate 80, a poly alkylene-oxide block polymer, such as pluronic PEO-PPO-PEO (PEO- polyethylene oxide, PPO- poly propylene oxide) pluronic R PPO-PEO-PPO, PBO-PEO (PBO- polybutylene oxide), tetronic, an oligomeric alkyl ethylene oxide, such as Brij or tergitol, an alkyl phenol polyethylene, such as Triton, and pluronic F-127. In some aspects, the surfactant of the cross-linking solution is of from about 0.2 wt% to 5 wt% of the polymer solution or the cross-linking solution, including about 0.2 to about 4.5 wt%, about 0.5 to about 4 wt%, about 0.8 to about 3.5 wt%, about 1.0 to about 3.0 wt%, and about 1.5 to about 2.5 wt%. In some aspects, when the cross-linking solution includes a surfactant, the polymer solution may further include a surfactant at a 3:4 ratio with respect to the carbonate compound.

[0087] In some aspects, the carbonate compound of the polymer solution includes calcium. In some aspects, the cross-linking solution includes calcium. It is an aspect of the present disclosure to provide calcium in an acidic environment to cross-link the polymer solution. It is an aspect of the present disclosure that the monomeric units within the polymer solution begin to cross-link upon contact with calcium ions in low pH. In some aspects, the cross-linking solution further includes a calcium salt, such as calcium chloride. In some aspects, the calcium can be of about 0 to about 8 wt% calcium chloride, including about 1 to about 7, about to about 6, about 1 to about 5 about 1 to about 4, about 1 to about 3, about 2 to about 7, about 2 to about 6, about 2 to about 4, about 3 to about 7, about 3 to about 6, about 3 to about 5 and about 5 to about 7 wt%. It will be appreciated that in some aspects, the crosslinking solution provides calcium to the cross-linking reaction, such as with calcium carbonate. In some aspects, calcium carbonate is of about 0.5 to about 30 wt% of the polymer weight (e.g. 2 wt% PGA yields 0.01 to 0.6 wt% CaCCh), including about 1 to about 25, about 5 to about 20, about 10 to about 20, about 10 to about 15, about 15 to about 25, and about 20 to about 25 wt% of the polymer weight. In some aspects, the cross-linking solution includes calcium and a surfactant. In some aspects, the surfactant is present at a ratio of 3:4 with respect to calcium in the cross-linking solution.

[0088] In aspects, the cross-linking solution includes an acid to provide the crosslinking solution with a pH less than or equal to 7, such as less than or equal to 2, less than or equal to 3, less than or equal to 4, less than or equal to 5, or less than or equal to 6. In some aspects, the acid is chosen from acetic acid and hydrochloric acid. In some aspects, the acid is about 0.5 to about 30 wt% of the cross-linking solution, including about 1 to about 25 wt%, about 5 to about 20 wt%, about 7 to about 15 wt%, and about 8 to about 12 wt%. In some aspects, the acid is about 10 wt% of the cross-linking solution.

[0089] In aspects, the methods of the present disclosure may also include stirring the dropped polymer solution in the cross-linking solution for a period of from about 5 minutes to about 10 hours, including about 10 to about 500 mins, about 30 to about 400 mins, about 60 to about 300 mins, about 90 to about 200, and about 120 to about 180 mins. In some aspects, the period is of about 10, 20, 30, 40, 50, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, 360, 390, 420, 450, 480, 510, 540, 570, and 600 minutes.

[0090] In some aspects, each drop of the polymer solution in the cross-linking solution is of about 0.05 pL to 5 pL, including about 0.1 to about 4 pL, about 0.1 to about 3 pL, about 0.1 to about 2 pL, about 0.1 to about 1 pL, about 0.5 to about 4 pL, about 0.5 to about 3 pL,about 0.5 to about 2 pL, about 0.5 to about 1 pL, about 1 to about 4 pL, about 1 to about 3 pL, about 1 to about 2 pL, about 1 to about 1 pL, about 2 to about 4 pL, about 2 to about 3 pL, and about 3 to about 4 pL,.

[0091] In some aspects, the microcarriers form as the calcium and carbonate interact with the polymer units in the acidic environment. The carbonate dissociates into CO2 gas and the calcium initiates cross-linking between suspended units in the polymer. The result is the added drop coalescing into a sphere or spheroid that forms with gas trapped therein. The trapped gas results in pores within. Controlling the size of the added drop can therefore provide a particular sized microcarrier, such as a from about 500 pm to about 2 mm, including about 750 pm to about 1500 pm, about 1000 pm to about 1500 pm, about 1250 pm to about 1500 pm, about 750 pm to about 1250 pm, about 1000 pm to about 1250 pm, about 500 pm to about 1500 pm, about 500 pm to about 1250 pm, about 500 pm to about 1000 pm, and about 500 pm to about 1250 pm.

[0092] In aspects, the methods of the present disclosure may also include collecting one or more formed microcarriers from the cross-linking solution. In some aspects, the collected microcarriers can then be dried. In some aspects, the collected microcarriers are lyophilized. In some aspects, the collected microcarriers are frozen or freeze-dried. In some aspects, the collected microcarriers are lyophilized and freeze-dried. Drying and / or freezing the microcarriers embeds the pore structures within the microcarrier beads allows the trapped gas to sublime the gas pockets.

[0093] In aspects, the methods of the present disclosure include exposing the internal pores within the formed and collected microcarriers or beads. In aspects, the internal pores of each microcarrier are largely covered during the polymerization in the cross-linking solution (see, e.g., FIG. 2a). The internal pores of the microcarrier may be exposed by removing all or a portion of the exterior surface of each bead. When the microcarriers are initially formed, the pores are inaccessible to cells as they do not connect to the surface. Therefore, the methods of the present disclosure may include removing the surface layer to reveal the porous interior (i.e., exfoliating the formed microcarriers or beads) by leveraging the same process used to dissolve the microcarriers albeit at a lower concentration and / or shorter time period (e.g., exposure to EDTA and pectinase). The exfoliation steps of the methods of the present disclosure have the benefit of not being limited to any specific chemistry and create a large design space to optimize the porosity, pore size distribution, and mechanical strength of the bead, though increased surface porosity may not be ideal for sufficiently protecting the cells from shearing.

[0094] In aspects, the exfoliating solution includes pectinase and a chelator. In some aspects, the exfoliating solution is aqueous. In some aspects, the chelator is EDTA (ethylenediaminetetraacetic acid). A sodium salt of ethylene glycol tetraacetic acid (EGTA), an acidic form of EGTA, and / or citric acid. In some aspects, the chelator is EDTA. In some aspects, the chelator is present at about 1 mM to about 20 mM. In some aspects, the pectinase is present with an activity of about 10 to about 200 U / mL, including about 20 to about 150 U / mL, about 50 to about 150 U / mL, about 100 to about 150 U / mL, about 20 to about 100 U / mL, and about 50 to about 100 U / mL. In certain aspects, the pectinase is present at about 50 U / mL. As is described herein, the exfoliating solution is capable of dissolving the entire microcarrier, and as such, exposure to such should be limited to allow for removal of the surface of the microcarrier only. While concentrations of the exfoliating solution and exposure times may vary, high concentrations and / or long exposure times may cause excessive deterioration of the microcarrier itself. As such, exposure should be for a limited period, such as less than 5 minutes, including less than or equal to 4, less than or equal to 3, less than or equal to 2, less than or equal to 1, less than or equal to 0.9, less than or equal to 0.8, less than or equal to 0.7, less than or equal to 0.6, less than or equal to 0.5, less than or equal to 0.4, less than or equal to 0.3, less than or equal to 0.2, and less than or equal to 0. 1 minutes.

[0095] Eollowing removal of the outer surface of the microcarrier(s) with the exfoliating solution, the methods of the present disclosure may then include washing the microcarrier(s) to remove further exfoliating solution.

[0096] In aspects, the internal porosity of the formed exfoliated porous microcarriers is greater than or equal to 90% of a volume of the porous bead. In some aspects, the porous bead has a diameter greater than or equal to 500 pm to less than or equal to 2 mm, including about 750 pm to about 1500 pm, about 1000 pm to about 1500 pm, about 1250 pm to about 1500 pm, about 750 pm to about 1250 pm, about 1000 pm to about 1250 pm, about 500 pm to about 1500 pm, about 500 pm to about 1250 pm, about 500 pm to about 1000 pm, and about 500 pm to about 1250 pm. In some aspects, the internal porosity includes pores with each pore having a diameter greater than or equal to 50 pm to less than 500 pm, including about 100 pm to about 400 pm, about 200 pm to about 400 pm, about 300 pm to about 400 pm, about 100 pm to about 300 pm, about 200 pm to about 300 pm, and about 100 pm to about 200 pm.

[0097] In some aspects, the microcarriers can then be cultured with one or more cells or cell types. It is an aspect of the present disclosure that cells can reside within the pores of the microcarriers, thereby providing a protected environment. Lor example, industrialapplications can require exposure to stressors such as force, shear stress, high flow, crude and rapid mixing, and so forth, all of which can damage a cell. The presence of the cells within the pores of the microcarriers provides a shelter from external stressors, thereby reducing damage and / or loss of cells in such environments. The cells can be incorporated within the microcarriers by gentle mixing and / or incubation. In some aspects, it may be beneficial to enzymatically treat cells to reduce adherence to allow for even distribution within the microcarriers. It will also be appreciated that the cells need not adhere in order to reside within the pores of the microcarriers.

[0098] It is a further aspect of the present disclosure that the microcarrier(s) of the present disclosure are dissolvable. Such can be particularly useful to release cultured cells without causing harm to the cells themselves. Increasing the concentration of the pectinase and / or chelator as well as increasing exposure time will allow for the microcarriers to dissolve and thereby release cells into the solution therein. For example, culturing cells on an industrial scale can expose the cells to stressors. The microcarriers offer shelter from such, but at some point will require harvesting. The aspect that the microcarriers are dissolvable allows for the cells to be retrieved from the microcarriers without any harsh treatment or exposure to digestive enzymes, thereby allowing a high recovery rate of cells. In short, dissolving the microcarriers releases the cells into the dissolving solution. As described herein, the dissolving solution may be the same as the exfoliating solution or a stronger or more active version of the exfoliating solution. The cells can then be collected from the dissolving solution, such as with simple centrifugation.

[0099] It is also a further aspect of the present disclosure to incubate exfoliated microcarriers with cells, such as by contacting the exfoliated microcarriers with a cell or introducing a cell suspension into a solution with exfoliated microcarriers. To encourage cell population into the pores of the microcarriers, the solution may be stirred or agitated. It is an aspect of the present disclosure that maintaining cells within the pores of the microcarriers helps to protect the cells from exposure to shear during any mechanical mixing. This in turn provides for culturing cells on a large (industrial) scale if desired. Dissolvable porous microcarriers are a universal platform for large scale cell culture and can be used broadly in a stirred tank, fluidized bed, and / or packaged bed. Porous microcarriers can support the culturing of strongly adherent cells, weakly adherent cells, and cells in suspension. Similarly, the dissolvability helps to address harvesting issues on a large scale and reliance on animal content that currently challenges other attempts to scale cell culture. Further, the dissolvable porousmicrocarriers can support using the cultured cells for production of other biological materials, such as nucleic acids, proteins, and even viruses or viral vectors.

[0100] In some aspects, the methods may further include contacting the exfoliated microcarriers, optionally with cells adhered or suspended therein, with a dissolving solution of a chealtor and pectinase. In some aspects, the methods may include collecting and / or isolating the cells from the dissolving solution.

[0101] In some aspects, the methods of the present disclosure include culturing cells within the porous microcarriers of the present disclosure. Such may including incubating the exfoliated porous microcarriers with cells adhered and / or suspended therein in a cell culture media. Such may include stirring and / or mixing of the microcarriers with cells in a solution. It will be appreciated that agitation or mixing of the cells in solution with the microcarriers should be of sufficient velocity to allow for the cells in solution to disperse evenly, yet of a velocity below which damage to the cells is likely. In some aspect, the agitation or mixing disperses the cells and microcarriers evenly in the solution. As cells come into contact with the microcarriers, they can be lodged or entrapped within the pores thereof. In some aspects, a user may be interested in the collection of one or more products from the cells. In such aspects, it is a further facet of the present disclosure wherein the cell culture media can be removed or exchanged and cell products collected therefrom. For example, if a secreted product, such as a gene or protein is to be collected, the media can be readily separated by collecting the microcarriers and isolating, such as through gravitational separation or by an applied force, such as centrifugation. In some aspects, the microcarriers can be re-suspended in fresh media. In other aspects, the microcarriers with the cells suspended and / or adhered therein can be placed in the exfoliation solution as described herein, which in effect acts as a digestion solution to dissolve the microcarrier entirely or at least to the point where the cells are therefrom. It will be apparent that gravity or a gravitational force, such as centrifugation can collect cells post digestion of the microcarriers.

[0102] In aspects, the present disclosure relates to cellular microcarrier(s) produced by the methods set forth herein.

[0103] In aspects, the present disclosure relates to a cellular microcarrier of porous (exfoliated) beads, wherein each bead comprises PGA cross-linked through calcium. Each bead contains pores therein. In some aspects, the pores are of about 50 pm to about 500 pm in diameter, including about 100 pm to about 400 pm, about 200 pm to about 400 pm, about 300pm to about 400 pm, about 100 pm to about 300 pm, about 200 pm to about 300 pm, and about 100 pm to about 200 pm. In some aspects, each porous bead is of about 500 pm to about 2 mm in diameter, including about 750 pm to about 1500 pm, about 1000 pm to about 1500 pm, about 1250 pm to about 1500 pm, about 750 pm to about 1250 pm, about 1000 pm to about 1250 pm, about 500 pm to about 1500 pm, about 500 pm to about 1250 pm, about 500 pm to about 1000 pm, and about 500 pm to about 1250 pm. In some aspects, the surface of each porous bead is exfoliated. In some aspects, the surface of each porous bead is exfoliated by contact with a solution comprised of pectinase and a chelator, as described herein. In some aspects, the pores of each porous bead are exposed on the surface. In some aspects, each porous bead may further include at least one cell adhered and / or suspended therein. Cells can be a mammalian cell, a bacterial cell, and / or an insect cell. In some aspects, the porous beads are exfoliated by an exfoliation solution. In some aspects, the exfoliation solutions is the same or weaker than a digestion solution. In some aspects, the exfoliation occurs through partial digestion of the bead. In some aspects, time and / or concentration of the digestion solution can allow for partial digestion and the exposure of the underlying pores. In some aspects, the exfoliation solution may be equal to or less than the concentration of the digestion solution, including about l / 10thl / 9th, l / 8th, l / 7th, l / 6th, l / 5th, l / 4th, l / 3rd, iri, 2 / 3rd,3 / 4, 7 / 8th, or 9 / 10thof the concentration of the digestion solution.

[0104] In aspects, the present disclosure relates to a cellular microcarrier of porous beads, wherein each bead is of PGA cross-linked through calcium with pores therein, wherein the pores are of about 50 pm to about 500 pm in diameter, including about 100 pm to about 400 pm, about 200 pm to about 400 pm, about 300 pm to about 400 pm, about 100 pm to about 300 pm, about 200 pm to about 300 pm, and about 100 pm to about 200 pm, wherein each porous bead is of about 500 pm to about 2 mm in diameter, including about 750 pm to about 1500 pm, about 1000 pm to about 1500 pm, about 1250 pm to about 1500 pm, about 750 pm to about 1250 pm, about 1000 pm to about 1250 pm, about 500 pm to about 1500 pm, about 500 pm to about 1250 pm, about 500 pm to about 1000 pm, and about 500 pm to about 1250 pm, and wherein a cell is adhered and / or suspended within a pore.EXAMPLES

[0105] The embodiments described herein will be further clarified by the following examples.

[0106] Two methods of synthesizing porous microcarriers were investigated: first, PGA and CaCh concentrations were varied in an external gelation process to introduce porosity with lower PGA concentrations. The second method used carbonates in the PGA solution and acid in the curing cross-linking solution. The reaction of the two solutions produces carbon dioxide which acts as a gassing agent to create internal bubbles as pore templates while polymer gelation occurs around these templates. With this method, carbonate / acid / calcium sources and concentrations were varied, along with the presence and concentration of surfactant, drop size, pre-mixing time, and drying techniques.

[0107] For the first experiment, introducing pores into the microcarrier structure by an external gelation method was performed, with systematic variance of the concentrations of PGA in the inlet solution and CaCh in the curing solution. The resultant structures were imaged scanning electron microscopy; samples are shown in FIG. la, lb, and 1c.

[0108] The theory in this approach was that reducing the concentration of the polymer may make a porous bead. CaCh concentration was also varied to determine if more stability might be helpful at lower concentrations, or if lowering the CaCh also adds porosity.

[0109] Briefly, water was used to make dilutions of PGA: 0.5, 1, 1.5, 2 wt% (50 mb each) and to make dilutions of CaCh + Tween 80 (CaCh:Tween 80 = 4:3) at the following concentrations of calcium chloride: 1, 2, 4, 6, 8 wt% (400 mb each). 100 mb of CaCh solution was added to a shallow dish with a stir bar on 300 RPM. An eye drop setup (tube hanging with clamp) was arranged and the desired flowrate established. Then PGA was added to the CaCh solution in a dropwise-manner. Drops of the PGA were added until the surface was approximately saturated with beads. The beads were then put in -80 °C freezer for 1-2 hours to lyophilize.

[0110] The dried beads were delicate. There was also significant diversity in quantitative quality within a single batch. Surface images of some beads appeared ruffled. No significant pores were observed on the surface. Upon sectioning the beads, pores were observed internally.

[0111] While varying PGA concentration had a seemingly small effect on porosity or structural integrity, varying CaCh concentration effected both porosity and structural integrity. At lower concentrations of CaCh, the microcarriers had larger pores; however, these pores were all internal as depicted in FIGS, lb and 1c. The surface of the beads were mostly smooth, indicating a perfectly gelled exterior with any internal porosity closed from cell entry. Somestructures on the surface showed ruffled-like motifs, which could potentially indicate collapsed pores during drying (FIG. la).

[0112] Further experiments were then conducted to investigate pore formation with gas foaming agents. In these experiments, a suspension of PGA and a carbonate foaming agent carbonate was dripped as spherical particles into a solution of calcium chloride, surfactant, and acid. The calcium ions in this curing solution acted as cross-linking agents to cure the polymer, while the acid reacted with the carbonate foaming agent to evolve gas bubbles. An example reaction with calcium carbonate and acetic acid to release carbon dioxide is given below:CaC02+ 2CthCOOH Cc + 2CH2COO- + H2CO3H2O + CO2

[0113] Since the polymer was cross-linked around these gas bubbles, the physical release of the bubbles resulted in pore formation in the polymer. This was accomplished by freezing the structures and subsequently lyophilizing them to sublime the frozen gas pockets; the exiting gas creates random channels and directs the porosity. The structures can also be directly lyophilized to force evacuation of gaseous CO2, forming a similar pore network. SEM images of bead cross-sections in FIGS. 2a, 2b, and 2c depict the resulting pore network. While little qualitative difference was observed between samples of varying calcium carbonate content, residence time in acid made a an impact on microcarrier formation. Samples that were left for an hour showed crystal residue on the surface, indicating that the CO2 evolution reaction had not proceeded to completion; this was not the case for samples left for greater time periods which did not exhibit crystallization near the surface.

[0114] Experiment 2

[0115] CaCCE reacts with acetic acid when PGA / CO3 drops and hits the CaCh / acid solution, forming CO2 as a gassing agent and creating bubbles. Freeze drying the samples preserves the structure as the frozen CO2 sublimes and tunnels through the gel to exit, creating a network of pores leading to the surface. In the experiment, CaCCE concentration was varied, as was the time in acid.

[0116] Three PGA / carbonate / water solutions (2 wt% PGA and 5, 10, and 20 wt% CaCCE, with water, 50 mb each) were prepared. A high speed mixer was used to suspend each of the 3 carbonate solutions before adding the carbonate solution to PGA / water at approximately 10,000 RPM for approximately 30 minutes. 400 mb of 4 wt% CaCh + 3 wt%Tween 80 and 10 wt% acetic acid (using glacial acetic acid) was also batched for the PGA solution to be added in later drop-wise.

[0117] 100 mL of the CaCh / Tween / acid solution was added to a shallow dish with a stir bar spinning at 300 RPM. After mixing PGA / carbonate solution using the high speed mixer, the PGA / carbonate solution was added to the CaCh / Tween / acid solution using a disposable dropper.

[0118] Thereafter, the resulting microcarriers were examined and bubbles inside of the beads were visible. Samples were section and SEM images of the samples were taken. It was confirmed that pores developed inside the microcarriers, but only tiny holes (if any) were on the surface. There were no noticeable differences between samples formed from different solution concentrations based on SEM images.

[0119] The surface of all the beads was generally smooth. Small openings were observed on the surface of each bead.

[0120] Without wishing to be bound by theory, it is believed that the lack of surface porosity may be attributed to the large diffusion length for a majority of the CO2 trapped inside the bead. That is, most of the gas does not reach the surface of the bead upon drying and remains trapped inside.

[0121] Experiment 3

[0122] An experiment was designed to decrease the bead diameter and thereby reduce bubble travel distance to the surface of the bead. As with Experiment 2, 5 wt% CaCCh was used and a smaller pipette tip employed to make smaller beads.

[0123] Visually, smaller beads resulted in better surface porosity as more small openings were visible in the resulting beads. Additionally, though the porosity measurements from mercury intrusion porosimetry were very high for all the samples, it was determined that a statistically significant difference was present in the porosity between two samples of the same composition and processing that varied only in size. Specifically, smaller beads had porosities of approximately 97%, comparable with most of the other samples that had small diameters and other varying conditions, while larger beads had a porosity of approximately 88%. Thus, the effect of a smaller bead diameter to minimize diffusion lengths of the acid inwards and the bubbles outward did have a noticeable improvement in surface porosity.

[0124] To achieve the desired pore morphology, the reaction speed between PGA crosslinking and gas formation can be balanced by controlling the reaction rates by altering the relative concentrations of the species and the timing of their availability. First, the ratio was reduced by reducing the PGA crosslinking rate. Specifically, the CO2 evolution reaction also releases calcium from the calcium carbonate. Rather than using a second CaCh solution as the source of Ca2+, the Ca2+already present in CaCCh (that is released as CO2 in the gas evolution reaction) can be used to cross-link the polymer. This also requires the gas evolution to commence before gelation can begin.

[0125] Another way to reduce the ratio of reaction rates is to increase the rate of gas generation. This strategy was followed in two separate ways. First, the acetic acid in the curing solution was replaced with hydrochloric acid. Because HC1 is a significantly stronger acid than AcOH, it increases the concentration of protons in solution and increases gas production. Second, a more dissociative salt was used as the CO2 source. By using sodium bicarbonate (dissolvable and thus already dissociated) rather than calcium carbonate (which requires the presence of acid to dissociate), gas was increased by instead increasing the concentration of real carbonate ions in solution.

[0126] After replacing the acetic acid with hydrochloric acid in experiment 5, the resulting microcarriers had bigger internal pores and very few surface pores (FIG. 3a and 3b). The large bubbles are believed to be due to the coalescence of smaller bubbles. Thus, surfactant was added directly to the PGA solution and continued throughout the rest of the experiments (FIG. 3c and 3d).

[0127] To reduce crosslinking rate, the calcium chloride was removed from curing solution and the calcium carbonate in the beads formulation was used as both the foaming agent and the cross-linking source. A stronger acid was needed to increase the rate to be competitive with CO2 evolution. Optical images showed it had a high density of bubbles, especially near the surface (FIG. 4d). SEM imaging confirmed that this method produced an improved amount of surface and interior pores of appropriate sizes (FIG. 4a, 4b, and 4c).

[0128] Experiment 4

[0129] Next, CaCh was removed all together from curing solution and the CaCCh was therefore the calcium source (with stronger acid) to make the processes of gelation and bubbling simultaneous so bubbles can reach the surface to affect the porosity of the surface asit forms. Also added surfactant to the PGA solution so it can break up bubbles (could not diffuse into the bead in time when present only in the acid solution).

[0130] Briefly, 100 mb of 2 wt% PGA in 5 wt% CaCCh (20 micron) was made as done in Experiment 2. Drops into 100 m of solutions described below were done with disposable eye dropper or other pipette tipped droppers to vary drop size, (whenever there is Tween 80 + CaC12, the CaC12:Tween weight percent ratio is always 4:3, because it is made from 8 wt% CaC12 6 wt% Tween 80 stock). Samples were then left in HC1 for at least a few hours, followed by freeze drying and lyophilization overnight.

[0131] Calcium carbonate is sufficient as a carbonate source to make spherical beads and bubbles were especially close to the surface. Further the presence / concentration of surfactant in both the PGA solution and the acid solution has an effect on the bead porosity and structure

[0132] Finally, the method to dissolve the microcarriers was leveraged as a method to exfoliate the outer shell and reveal the internal pores. By exposing the synthesized beads to a solution with EDTA and pectinase, the outer surface could be consumed retroactively. Using the same concentrations used to dissolve DMCs and a range of short exposure times was tested. C aCCh as the foaming agent dripped into HC1. The SEM images from experiment 5 below show the evolution of the surface structure with time (FIG. 5). Exposing the microcarriers to a solution of EDTA and pectinase successfully acted as an exfoliant to remove the outer layer, revealing the desirable porous structure.

[0133] Experiment 5

[0134] Beads from experiment 4 were taken and exfoliated with 5 mM EDTA / 50U / ml pectinase / PBS solution. The beads were then left in the solution for various amounts of time.

[0135] Briefly, a solution of 5 mM EDTA / 50U / ml pectinase / PBS was prepared and warmed to over to ~50 °C. Some wet beads from experiment 4 (not dried) were obtained and dropped in solution. They were then stirred and samples were subsequently removed at 15, 30, 45, 60, 90 and 120 seconds. Samples were rinsed with water immediately upon removing from the solution and then put straight into the lyophilizer (no freezing).

[0136] Experiment 6

[0137] This experiment was to use a drop mix of PGA / F-127 / NaHCC>3 into water / acetic acid / CaCh. (using more quickly dissociating carbonate instead of stronger acid).

[0138] Briefly, 100 mL of 2 wt% PGA + 1 wt% F-127 + 0.9 wt% sodium bicarbonate was made and then stirred at -10,000 RPM for -30 mins (stirring just below surface to prevent incorporation of ambient air). At least 100 mL of 10 v% acetic acid + 0.1 M CaC12 with a dropper with pipette tip was then added (medium sized beads, no stirring). The beads were left in acid overnight, followed by overnight lyophilization.

[0139] Optical images showed a lot of small, well distributed bubbles and SEM showed good surface pores.

[0140] Microcarriers offer a surface for adhesion during incubation on which a greater number of cells can be cultured. Some cell strains are sensitive to the shear created by bioreactors, making porous microcarriers that protect these cells within the microcarrier structure desirable. However, to separate the cultured cells from the microcarriers, agitation is required, which can also harm shear sensitive cells and reduce yield. To solve this problem, this work was to synthesize porous dissolvable microcarriers.

[0141] Several promising syntheses have been identified to produce viable structures. The first method replaces CaCCL with NaHCCfi among other changes to produce a porous dissolvable microcarrier suitable for cell culture. The second method allows the use of a wide variety of process conditions with an additional exfoliation step to tune the surface of the microcarrier as necessary.

[0142] It will be apparent to those skilled in the art that various modifications and variations can be made to the embodiments described herein without departing from the spirit and scope of the claimed subject matter. Thus it is intended that the specification cover the modifications and variations of the various embodiments described herein provided such modification and variations come within the scope of the appended claims and their equivalents.

[0143] It is appreciated that all reagents are obtainable by sources known in the art unless otherwise specified.

[0144] It is also to be understood that this disclosure is not limited to the specific aspects and methods described herein, as specific components and / or conditions may, of course, vary. Furthermore, the terminology used herein is used only for the purpose of describing particular aspects of the present disclosure and is not intended to be limiting in any way. It will be also understood that, although the terms “first,” “second,” “third” etc. may be used herein to describe various elements, components, regions, layers, and / or sections, theseelements, components, regions, layers, and / or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer, or section from another element, component, region, layer, or section. Thus, “a first element,” “component,” “region,” “layer,” or “section” discussed below could be termed a second (or other) element, component, region, layer, or section without departing from the teachings herein. Similarly, as used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms, including “at least one,” unless the content clearly indicates otherwise. “Or” means “and / or.” As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items. It will be further understood that the terms “comprises” and / or “comprising,” or “includes” and / or “including” when used in this specification, specify the presence of stated features, regions, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and / or groups thereof. The term “or a combination thereof’ means a combination including at least one of the foregoing elements.

[0145] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It will be further understood that terms such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure, and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0146] Reference is made in detail to exemplary compositions, aspects and methods of the present disclosure, which constitute the best modes of practicing the disclosure presently known to the inventors. The Figures are not necessarily to scale. However, it is to be understood that the disclosed aspects are merely exemplary of the disclosure that may be embodied in various and alternative forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for any aspect of the disclosure and / or as a representative basis for teaching one skilled in the art to variously employ the present disclosure.

[0147] Patents, publications, and applications mentioned in the specification are indicative of the levels of those skilled in the art to which the disclosure pertains . These patents, publications, and applications are incorporated herein by reference to the same extent as if each individual patent, publication, or application was specifically and individually incorporated herein by reference.The foregoing description is illustrative of particular embodiments of the disclosure, but is not meant to be a limitation upon the practice thereof. The following claims, including all equivalents thereof, are intended to define the scope of the disclosure.

Claims

CLAIMSWe claim:

1. A method for producing porous beads for use as cellular microcarriers, the method comprising: adding, drop wise, a polymer solution comprised of a suspended polymer and a carbonate compound into a cross-linking solution comprising an acid, wherein each drop of the polymer solution reacts with the cross-linking solution to form a porous bead, the porous bead comprising an internal porosity; and treating the porous bead with an exfoliating solution to expose the internal porosity of the porous bead at a surface of the porous bead.

2. The method of claim 1, wherein the cross-linking solution is actively stirred during the adding.

3. The method of claim 1, wherein the polymer solution comprises a suspended pectic polymer.

4. The method of claim 3, wherein the suspended pectic polymer comprises polygalacturonic acid.

5. The method of claim 3, wherein the suspended pectic polymer comprises from about 0.1 wt% to about 5 wt% of the polymer solution.

6. The method of claim 5, wherein the suspended pectic polymer comprises 2 wt% of the polymer solution.

7. The method of claim 1, wherein the carbonate compound is selected from the group consisting of calcium carbonate and sodium bicarbonate.

8. The method of claim 1, wherein the carbonate compound comprises from 2 wt% to 30 wt% of the polymer solution.

9. The method of claim 8, wherein the carbonate compound comprises 20 wt% of the polymer solution.

10. The method of claim 1, wherein the polymer solution further comprises a surfactant.

11. The method of claim 10, wherein the surfactant is a non-ionic surfactant.

12. The method of claim 10, wherein the surfactant is selected from the group consisting of polysorbate 80 and pluronic F-127.

13. The method of claim 10, wherein the surfactant comprises from 0.2 wt% to 5 wt% of the polymer solution or the cross-linking solution.

14. The method of claim 10, wherein the polymer solution further comprises a surfactant at a 3:4 ratio with respect to the carbonate compound.

15. The method of claim 1, wherein the cross-linking solution comprises a surfactant.

16. The method of claim 15, wherein the surfactant is a non-ionic surfactant.

17. The method of claim 15, wherein the surfactant is selected from the group consisting of polysorbate 80 and pluronic F-127.

18. The method of claim 15, wherein the surfactant comprises from 0.2 wt% to 5 wt% of the polymer solution or the cross-linking solution.

19. The method of claim 15, wherein the polymer solution further comprises a surfactant at a 3:4 ratio with respect to the carbonate compound.

20. The method of claim 1, wherein the carbonate compound comprises calcium to crosslink the polymer solution.

21. The method of claim 1, wherein the carbonate compound comprises calcium carbonate at about 0.5 to about 30 wt% of the suspended polymer weight.

22. The method of claim 1, wherein the cross-linking solution further comprises a calcium salt.

23. The method of claim 22, wherein the calcium salt is calcium chloride.

24. The method of claim 23, wherein the calcium chloride comprises about 0 to about 8 wt% of the cross-linking solution.

25. The method of claim 23, wherein the cross-linking solution further comprises a surfactant.

26. The method of claim 25, wherein the surfactant is present at a ratio of 3:4 with respect to calcium in the cross-linking solution.

27. The method of claim 25, wherein the surfactant is a non-ionic surfactant.

28. The method of claim 25, wherein the surfactant is selected from the group consisting of polysorbate 80 and Pluronic f-127.

29. The method of claim 1, wherein the acid is selected from the group consisting of acetic acid and hydrochloric acid.

30. The method of claim 1, wherein the acid comprises about 10 wt% of the cross-linking solution.

31. The method of claim 1, further comprising stirring the dropped polymer solution in the cross-linking solution for a period of from about 5 minutes to about 10 hours.

32. The method of claim 1, wherein each drop comprises about 0.05 pL to about 5 pL.

33. The method of claim 1, further comprising collecting formed microcarriers from the cross-linking solution.

34. The method of claim 33, further comprising treating the microcarriers collected from the cross-linking solution with an exfoliating solution to form exfoliated microcarriers.

35. The method of claim 34, wherein the exfoliating solution comprises pectinase and a chelator.

36. The method of claim 35, wherein the chelator comprises EDTA (ethylenediaminetetraacetic acid), ethylene glycol tetraacetic acid (EGTA), citric acid, or a combination thereof..

37. The method of claim 35, wherein the chelator is present at about 1 mM to about 20 mM.

38. The method of claim 35, wherein the pectinase is present at about 10 to about 200 U / mL.

39. The method of claim 36, wherein the pectinase is present at 50 U / mL.

40. The method of claim 34, further comprising contacting the exfoliated microcarriers with a cell.

41. The method of claim 40, further comprising contacting the exfoliated microcarriers with a dissolving solution comprising a chelator and pectinase.

42. The method of claim 1, wherein the internal porosity is greater than or equal to 90% of a volume of the porous bead.

43. The method of claim 1 , wherein the porous bead has a diameter greater than or equal to 500 pm to less than or equal to 2 mm.

44. The method of claim 1, wherein the internal porosity comprises pores, each pore comprising a diameter greater than or equal to 50 pm to less than 500 pm.

45. A cellular microcarrier produced by the method of claim 1 .

46. A cellular microcarrier comprising porous beads, wherein each bead comprises PGA cross-linked through calcium and pores therein, wherein the pores are from about 50 pm to about 500 pm in diameter and wherein each porous bead is from about 500 pm to about 2 mm in diameter.

47. The cellular microcarrier of claim 46, wherein a surface of each porous bead is exfoliated.

48. The cellular microcarrier of claim 47, wherein the surface of each porous bead is exfoliated by contact with a solution comprising pectinase and a chelator.

49. The cellular microcarrier of claim 48, wherein the pores of each porous bead are exposed on the surface.

50. The cellular microcarrier of claim 49, further comprising a cell.

51. The cellular microcarrier of claim 50, wherein the cell is selected from the group consisting of a mammalian cell, a bacterial cell, and an insect cell.

52. A cellular microcarrier comprising porous beads, wherein each bead comprises PGA cross-linked through calcium and pores therein, wherein the pores are of about 50 pm to about 500 pm in diameter, wherein each porous bead is from about 500 pm to about 2 mm in diameter, and wherein a cell is adhered within a pore.

53. The cellular microcarrier of claim 52, wherein the cell is selected from the group consisting of a mammalian cell, a bacterial cell, and an insect cell.

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