Bioink containing self-organizing peptides

Abstract

The bioink comprises a cell solution containing live cells and a cell culture medium, and a self-assembling peptide solution containing a self-assembling peptide. The bioink is formed by mixing and extruding both the cell solution and the self-assembling peptide solution, and after mixing, the bioink can be continuously extruded from a mixer. Also disclosed is a useful method for producing the bioink. It is apparent to those skilled in the art that the present invention brings about an important advancement in the technology of bioink.

Description

Technical Field

[0001] (Priority) This application claims priority to U.S. Provisional Application No. 63 / 327,825, filed on April 6, 2022, the entire content of which is incorporated herein by reference.

[0002] (Sequence Listing) This application includes an XML sequence listing that was electronically submitted and is incorporated herein by reference in its entirety. The sequence listing created on March 20, 2023, is named 3DM-21-02-3DP1-US-SL.xml and is 30,428 bytes in size.

[0003] (Field of the Invention) The present invention relates to a bioink suitable for 3D printing, and more specifically, to an improved bioink containing self-assembling peptides.

Background Art

[0004] (Background of the Invention) 3D printing of tissues involves the assembly of cells into a controlled volume, with the bioink mimicking the in vivo characteristics of the tissue. Such bioinks have competing design constraints. For example, if a particular shape is desired, the material used to create that shape must not only be extrudable but also ultimately be able to retain that shape. However, the addition of the requirement of live cells means that the materials used and the way the materials are extruded must be non-toxic to live cells. Various attempts to create bioinks are known. These include seeding cells onto the printed structure, mixing cells with the printing medium and then printing them into the desired structure, and printing cell clusters (spheroids) and arranging them together to form a functional tissue. Since this technology is quite new, various printers and printing techniques for printing cells and matrices have been developed. Commonly used methods of bioprinting are extrusion, laser, inkjet, and tissue fragment printing, all of which aim to place live cells and / or biomaterials and create a functional tissue analogue.

[0005] For example, many different materials are used as bioinks, including natural materials such as alginates, gelatin, collagen, silk, gellan gum, hyaluronic acid, dextran, and cellulose; synthetic materials such as polycaprolactone, Pluronic® acid, and polyethylene glycol; and commercial materials such as Derma Matrix®, Novogel®, and CELLINK®. The bioink should be printable with easy and cell-friendly crosslinking after printing / extrusion so as to maintain firmness immediately after extrusion, should hold its shape so as to accurately mimic the desired shape, should be biocompatible and biodegradable so as to not only allow cells to live but also enable cell growth and not cause an immune reaction. Further, such a bioink should preferably mimic the natural tissue environment to which cells attach and / or grow, and such a bioink should be customizable so as to be modifiable for various applications (such as soft tissue, middle tissue, or hard tissue). Despite numerous attempts at the advancement of bioprinting technology, the development of a satisfactory bioink that meets all the requirements for creating a biomaterial that serves as a suitable functional tissue analog is limited. It is desirable to provide a bioink that more satisfactorily meets these competing design constraints. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0006] (SUMMARY OF THE INVENTION) According to a first aspect, the bioink comprises a cell solution containing live cells and a cell culture medium, and a self-assembling peptide solution containing a self-assembling peptide. The bioink is formed by mixing the cell solution and the self-assembling peptide solution together and extruding them, and the bioink is extruded from a mixer after mixing. Also, a useful method for making the bioink is disclosed and described in more detail below.

[0007] From the above disclosure and the more detailed description of the various embodiments below, it will be apparent to those skilled in the art that the present invention provides an important advancement in the technology of bioinks. Of particular importance in this regard is the potential of the present invention to provide high-quality bioinks that are easily extrudable, customizable, and non-toxic to living cells. Additional features and advantages of the various embodiments will be better understood from the detailed description provided below.

Mode for Carrying Out the Invention

[0008] (Detailed Description of Specific Embodiments) It will be apparent to those skilled in the art, i.e., those with knowledge or experience in this technical field, that many variations in the use and design of the bioinks disclosed herein are possible. The following detailed discussion of the various alternative features and embodiments will explain the general principles of the present invention with reference to the 3D printing of bioinks and, more specifically, the medical and research applications of such bioinks. Other embodiments suitable for other applications will be apparent to those skilled in the art in view of the advantages of the present disclosure.

[0009] According to a first aspect, there is provided a bioink formed from a combination of a self-assembling peptide solution and a cell solution. The self-assembling peptide solution is one of many self-assembling peptides, for example, RADA16, a 16-amino acid peptide having protecting groups at both ends, i.e., AcN-RADARADARADARADA-CNH 2) including. An example of a suitable commercially available self-assembling peptide solution is PuraStat® from 3-D Matrix Medical Technology, Inc., which is an aqueous solution of approximately 2.5% RADA16. RADA16 has alternating hydrophobic (alanine) groups and hydrophilic (arginine and aspartic acid) groups that enable organization into stable β-sheets. The resulting porous hydrogel contains a high water content (about 99%) and resembles the extracellular matrix of natural body tissues. Optionally, a sucrose solution can be incorporated into the self-assembling peptide solution. Other self-assembling peptides, including shorter RADA derivatives (e.g., RADA8, RADA12), peptide mimetics (e.g., D-forms of amino acids), peptide amphiphiles, other concentrations, and other self-assembling peptide solutions suitable for use in bioinks, will be readily apparent to those skilled in the art in view of the advantages of the present disclosure.

[0010] In some embodiments, the SAP comprises an amino acid residue sequence that conforms to one or more of Formulas I-IV:

Number

[0011] In some embodiments, the SAP further comprises an amino acid sequence that interacts with the extracellular matrix, and the amino acid sequence anchors the SAP to the extracellular matrix.

[0012] In some embodiments, the amino acid residues in the SAP can be naturally occurring amino acid residues or non-naturally occurring amino acid residues. Naturally occurring amino acids can include amino acid residues encoded by the standard genetic code, as well as non-standard amino acids (e.g., amino acids having a D-configuration instead of an L-configuration), and amino acids that can be formed by modification of standard amino acids (e.g., pyrrolysine or selenocysteine). Suitable non-naturally occurring amino acids can include, but are not limited to, D-alloisoleucine (2R,3S)-2-amino-3-methylpentanoic acid, L-cyclopentylglycine (S)-2-amino-2-cyclopentylacetic acid.

[0013] In some embodiments, the specific peptides for use in the methods of the invention can be selected from one or more of the peptides listed in Table 1 below. [Table 1]

[0014] In other embodiments, another class of materials that can self-organize are peptidomimetics. As used herein, peptidomimetics refer to molecules that mimic the peptide structure. Peptidomimetics have general characteristics similar to those of the polypeptides that are their parent structures, such as amphiphilicity. Examples of such peptidomimetic materials are described in Moore et al., Chem. Rev. 101(12), 3893-4012 (2001). Peptidomimetic materials can be classified into four categories: α-peptides, β-peptides, γ-peptides, and δ-peptides. Copolymers of these peptides can also be used. Examples of peptidomimetics of α-peptides include, but are not limited to, N,N′-linked oligoureas, oligopyrrolinones, oxazolidin-2-ones, azatides, and azapeptides. Examples of β-peptides include, but are not limited to, β-peptide foldamers, α-amino acids, sulfur-containing β-peptide analogs, and hydrazinopep tides. Examples of γ-peptides include, but are not limited to, γ-peptide foldamers, oligoureas, oligocarbamates, and phosphodiesters. Examples of δ-peptides include, but are not limited to, alkene-based δ-amino acids and carbopeptoids such as pyranose-based carbopeptoids and furanose-based carbopeptoids.

[0015] In certain embodiments, the SAP is AC5®, AC5-V®, or AC5-G™, manufactured by Arch Therapeutics, Inc. (see www.archtherapeutics.com).

[0016] The cell solution may contain any one of several living cells, or a combination of living cells. There are numerous examples, including stromal vascular fraction (SVF) cells, stem cells, tumor cells, hepatocyte progenitor cells, rat pheochromocytoma cells (PC12), hippocampal neurons, endothelial cells, nerve cells, fibroblasts, keratinocytes, and transformed cells (including, for example, MG-62, SH-SY5Y, HEK293, NIH3T3). Other cells suitable for use in the cell solution of the bioink disclosed herein will be readily apparent to those skilled in the art in view of the advantages of the present disclosure.

[0017] The cell solution typically contains a cell culture medium for nourishing the cells and maintaining a healthy environment. Suitable components of the cell culture medium can include, for example, Dulbecco's Modified Eagle Medium (DMEM), a basal medium widely used to support the growth of many different mammalian cells, fetal bovine serum, Minimum Essential Medium (MEM), non-essential amino acids, an L-glutamine solution, antibiotics such as penicillin and streptomycin, and a trypsin-EDTA (ethylenediaminetetraacetic acid) solution. Other cell culture medium components suitable for use in the cell solution of the bioink disclosed herein will be readily apparent to those skilled in the art in view of the advantages of the present disclosure.

[0018] According to one embodiment, the bioink can be formed by combining a self-assembling peptide solution with a cell solution in a mixer. After mixing together to form the bioink, the bioink can be extruded. Preferably, this occurs immediately or continuously. Here, "continuously" is understood to mean that when the self-assembling peptide solution and / or the cell solution is added to the mixer, the bioink of the mixed material is forced or promoted to be extruded from the mixer. Of course, this assumes that the control valve of the nozzle of the extruder is open and the mixture flows out of the chamber through the nozzle. Usually, the time between mixing and extrusion is relatively short, such as within 1 minute after mixing, usually only a few seconds. That is, usually, the mixer / extruder can have a chamber where mixing occurs upon receiving both the self-assembling peptide solution and the cell solution, but the mixture only exists in the chamber for a short time before moving to the nozzle. Generally, when the pH of the self-assembling peptide solution increases, cross-linking occurs by mixing with the cell solution to form the bioink. It is preferable to extrude the bioink immediately so that most of the cross-linking occurs after the bioink is extruded and a formed object is formed. Optionally, a solidifying solution such as one containing NaOH can be added to assist in controlling the pH of the bioink. In the extruder, a ratio of 0.5 - 1.5 / 100 (such as NaOH 1N / RADA16, solidifying solution / self-assembling peptide solution) can be used, 1 / 100 (NaOH 1N / RADA16) is more preferable, and it is preferable to mix together. The solidifying solution can be filled into a 10 mL syringe under sterile conditions, for example, immediately before connecting to the mixing extruder. In normal operation, the self-assembling peptide solution and the cell solution (and any solidifying solution) are continuously introduced together and mixed, and by the act of introducing more cell solution and self-assembling peptide solution together, the mixed bioink is forced to be extruded through the nozzle into an object having a desired shape.Suitable examples of mixers can include, for example, the T333 traditional FDM ("Fused Deposition Modeling") 3D printer made by the French company Tobeca, provided with a mixing chamber for receiving both solutions and an adjacent 1 mm nozzle, and when more self-assembling peptide solution, cell solution, or either of the two is introduced, the bioink is immediately forced through the nozzle. The volume of the mixture can be controlled with location and can also be controlled simultaneously. Other mixers and 3D printers suitable for mixing and extruding bioink will be readily apparent to those skilled in the art in view of the advantages of the present disclosure. Optionally, the bioink can be at least partially immersed in an immersion solution after extrusion, providing additional nutrients that help maintain the viability of the cells within the bioink.

[0019] Typically, the ratio of self-assembling peptide solution to cell solution mixed together to form bioink is 6:1 to 20:1, 8:1 to 13:1, or about 10:1 by volume. Advantageously, the bioink, by cross-linking of the self-assembling peptide solution, creates an object with sufficient rigidity / viscosity for many applications and thus preferably need not contain any additional thickening agents. Further, the self-assembling peptide solution may be kept refrigerated or at room temperature (i.e., at least above 5°C, above 10°C, or above 15°C) before mixing with the cell solution and / or after mixing with the cell solution but before the step of extruding the bioink. The cell solution can be kept at the temperature at which the cells normally live, about 35 - 40°C for cells living in humans, more specifically, for example, around 37°C. The bioink can be a mixture of a self-assembling peptide solution and a cell solution and hardens to form an object having a desired shape or a pre-programmed shape. The object spontaneously hardens after extrusion without additional heating or other processing steps by a technician.

[0020] During any of the processes of mixing, extrusion, or both mixing and extrusion, the shear stress on the bioink is preferably kept below the viability limit of living cells, such as 4000 PA. The shear stress can be controlled by limiting the speed of mixing and / or extrusion. Also, the bioink can be immersed in an immersion solution after extrusion to assist and / or protect the cells present. The immersion solution can include a basal medium for supporting the growth of many different types of cells, particularly mammalian cells, such as Dulbecco's Modified Eagle Medium (DMEM). Other compositions and combinations of chemicals suitable for use as the immersion solution will be readily apparent to those skilled in the art in view of the advantages of the present disclosure.

Example

[0021] Example 1. An example of a mixed bioink is as follows. A 2.5% (w / v) RADA16 self-assembling peptide solution was filled into a mixing extruder such that the ratio of RADA16 to the cell solution was approximately 10:1 by volume. RADA16 was filled under sterile and ambient conditions immediately before connecting to the mixing extruder. The cell solution contained fibroblasts expressing green fluorescent protein (GFP) (NIH3T3 / GFP, AKR-214, Cell Biolabs Inc., USA), was grown and suspended in DMEM (high glucose) from Gibco (France), and supplemented with 10% (v / v) fetal bovine serum (FBS) from Gibco (France), 0.1 mM MEM non-essential amino acids (NEAA) from Invitrogen (France), 2 mM L-glutamine from Gibco (France), and 1% (w / v) penicillin / streptomycin (10,000 U / mL) from Gibco (France). Before introduction into the mixer / extruder, the cell solution was trypsinized (0.25% (v / v) trypsin-EDTA from ThermoFisher (France)) and counted. Approximately 2.3x10 7A cell solution having cells / mL was connected to a mixing extruder. After introducing a self-assembling peptide solution into the mixer together with the cell solution, the resulting bioink was extruded into a shaped object. After bioprinting, the shaped object was immersed in a dipping solution of 5 mL of DMEM (high glucose) from Gibco (France) supplemented with 10% (v / v) fetal bovine serum (FBS) from Gibco (France), 0.1 mM MEM non-essential amino acids (NEAA) from Invitrogen (France), 2 mM L-glutamine from Gibco (France), and 1% (w / v) penicillin / streptomycin (10,000 U / mL) from Gibco (France), and placed at 37 °C in a 5% CO2 incubator to keep the cells warm at this preferred temperature. NIH 3T3 eGFP mouse fibroblasts were maintained in this state and were observed to be alive even after 60 days. The maximum shear stress was measured to be 3063 PA. Another important observation is that the pH of the self-assembling peptide solution is immediately neutralized by the ions in the live cell solution during co-extrusion. This indicated that the cells were not affected by the initial acidic pH of the peptide. The structural integrity of the shaped object formed by the bioink was maintained.

[0022] From the above disclosure and the detailed description of the specific embodiments, it is clear that various modifications, additions, and other alternative embodiments are possible without departing from the true scope of the invention. The embodiments discussed have been selected and described in order to best illustrate the principles of the invention and its practical application, whereby those skilled in the art can use the invention by making various modifications to suit the various embodiments and the specific uses contemplated. All such modifications and variations are within the scope of the invention as determined by the appended claims when they are construed in accordance with the scope to which they are fairly, legally, and equitably entitled.

Claims

1. A cell solution comprising living cells and a cell culture medium; and An self-assembling peptide solution comprising an self-assembling peptide A bioink comprising a combination of wherein the bioink is formed by mixing and extruding the cell solution and the self-assembling peptide solution, and is extruded from a mixer after mixing.

2. The bioink according to claim 1, wherein the ratio of the self-assembling peptide solution to the cell solution is between 6:1 and 20:1 by volume.

3. The bioink according to claim 2, wherein the ratio is between 8:1 and 13:1 by volume.

4. The bioink according to claim 1, wherein the self-assembling peptide is selected from Table 1.

5. The bioink according to claim 1, wherein the bioink does not contain a thickening agent.

6. The bioink according to claim 1, wherein the introduction of the self-assembling peptide solution, one of the cell solutions, or both into the mixer promotes the extrusion of the bioink from the mixer.

7. The bioink according to claim 1, further comprising an immersion solution, wherein the bioink is at least partially immersed in the immersion solution.

8. The bioink according to claim 1, further comprising a solidifying solution for adjusting the pH added to the combination of the cell solution and the self-assembling peptide solution before mixing and extruding.

9. A method for producing a bioink, comprising Mixing a cell solution comprising living cells and a cell culture medium with a self-assembling peptide solution comprising a self-assembling peptide in a mixer to form a mixture; Extruding the mixture to form an object; and Hardening the object A method comprising a combination of.

10. The method for producing a bioink according to claim 9, wherein after the mixing step, the mixture is continuously extruded from the mixer.

11. The method for producing a bioink according to claim 9, wherein the hardening step occurs spontaneously after the step of mixing the cell solution with the self-assembling peptide solution.

12. The method according to claim 9, wherein the extruder defines a chamber adjacent to the nozzle, and the mixing step occurs within the chamber.

13. The method according to claim 9, wherein the extruding step comprises extruding the bioink through the nozzle. **Claim 14** The method according to claim 9, wherein during the mixing step and the extruding step, the shear stress on the bioink is maintained below the survival limit of the living cells. **Claim 15** The method according to claim 9, further comprising the step of immersing the object in an immersion solution. **Claim 16** The method according to claim 4, wherein the self-assembling peptide is RAD A16.

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