JP2022171601A - Controlling freeze-drying of hydrogel - Google Patents

Abstract

To provide a method for controlling freeze-drying and reconstitution of a hydrogel comprising nanoscale cellulose.SOLUTION: The present disclosure relates to a method for controlling freeze-drying of a hydrogel, where the method comprises adjusting the residual water content of the freeze-dried hydrogel. The method comprises adding a biomolecule to the hydrogel before freeze-drying the hydrogel, and/or optimizing the freeze-drying cycle, e.g., by taking into account Tg' (glass transition temperature of a maximally freeze-concentrated sample) of the mixture comprising the hydrogel and the biomolecule. The present disclosure further provides the use of a freeze-dried hydrogel composition for cell culture, a cell-culturing scaffold, and a method for manufacturing a reconstituted hydrogel.SELECTED DRAWING: None

Description

FIELD OF THE DISCLOSURE The present disclosure is a method of controlling lyophilization of a hydrogel comprising nanoscale cellulose, the method comprising adjusting the residual moisture content of the lyophilized hydrogel, and nanoscale cellulose, oligosaccharides and It relates to a lyophilized hydrogel composition comprising at least one biomolecule selected from disaccharides and preferably residual moisture. The present disclosure further relates to the use of lyophilized hydrogel compositions for cell culture, cell culture scaffolds, and methods of making reconstituted hydrogels.

BACKGROUND OF THE DISCLOSURE In recent years, cellulosic hydrogel nanomaterials with biodegradable and biocompatible characteristics have attracted interest for different biopharmaceutical applications. Cellulosic hydrogel nanomaterials are of particular interest based on their unique properties and similarity to physiological matrices.

Drying of, for example, wood-based natural nanofibrillar cellulose (NFC) hydrogels has been investigated to enable better transport feasibility of the material and to improve storage properties at room temperature.

Freeze-drying, a drying method widely used to improve the shelf life and handling of heat-sensitive products, is one method for preserving heat-sensitive substances. However, the hydrophilic nature of nanocellulose fibrils and their tendency to aggregate during the drying process presents obstacles to attempts to dry NFC hydrogels. In addition, freeze-drying sometimes damages the product and can change product properties during long-term storage.

Despite ongoing research and development in the preservation of cellulosic hydrogel nanomaterials, a need still exists to provide improved methods for the preservation of hydrogels containing nanoscale cellulose.

BRIEF DESCRIPTION OF THE DISCLOSURE It is an object of the present disclosure to provide a method of controlling lyophilization and reconstitution of hydrogels comprising nanoscale cellulose by adjusting the residual moisture content of the lyophilized hydrogel. In some embodiments of the present disclosure, the method includes adding biomolecules to the hydrogel prior to freeze-drying the hydrogel, and/or the Tg' of a mixture comprising, for example, the hydrogel and biomolecules (maximum freeze-concentration sample optimizing the freeze-drying cycle by considering the glass transition temperature). Typically, hydrogels comprise nanoscale cellulose, such as nanofibrillar cellulose, and at least one biomolecule selected from oligosaccharides or disaccharides.

The present disclosure provides methods for controlling and steering hydrogel freeze-drying, for example, to ensure successful reconstitution of freeze-dried hydrogels and to understand molecular-level interactions with water during freeze-drying. It is based on the idea of a desire to identify

An advantage of the methods and lyophilized hydrogel compositions of the present disclosure is that the lyophilized hydrogels can be stored at room temperature (20-25° C.) for long periods of time, even greater than a year, and retain the properties of the hydrogels used for lyophilization. It can be reconfigured while Lyophilized hydrogels can be reconstituted to a desired shape, concentration, and/or consistency.

A further advantage of the lyophilized hydrogel composition of the present disclosure is that it can be used for cell culture and in methods of manufacturing cell culture scaffolds. Since the hydrogel concentration optimized for cell culture is different for different cells, it is not clear that different concentrations of hydrogel can be used for lyophilization, and that lyophilized hydrogel can be reconstituted to its original concentration or to a different concentration. Advantageous.

A further advantage is that the temperature of primary drying can be significantly increased by the method of the present disclosure. This has a significant impact from a time consumption and economic point of view, since the time required for primary drying can be significantly reduced if the sample is already warmed by 1°C during freeze-drying.

The methods of the present disclosure are particularly suitable when hydrogels comprising nanoscale cellulose, such as native nanofibrillar cellulose, are freeze-dried, eg, for use in cell culture.

BRIEF DESCRIPTION OF THE FIGURES Hereinafter, the present disclosure will be described in more detail by way of preferred embodiments with reference to the accompanying drawings.

FIG. 1 shows nanofibrillar cellulose without biomolecule (control, A), with 300 mM sucrose (B), with 150 mM trehalose and 333 mM glycine (C), and with 300 mM trehalose (D). Figure 2 shows the shear rate viscosity of freeze-dried cakes from (NFC) hydrogel compositions before freeze-drying (FD) and after reconstitution (mean ± S.D., n=3); Figure 2 shows pre-lyophilized and reconstituted nanofibrillar cellulose (NFC) compositions without biomolecules (control, A), with 300 mM sucrose (B), and with 300 mM trehalose (C). Storage modulus (G′) and loss modulus (G″) after construction are shown (mean±SD, n=3); Figure 3 shows 3D viability of HepG2 cell spheroids (A) and PC3 cell spheroids (B) cultured in reconstituted lyophilized (FD) hydrogels and fresh non-lyophilized hydrogels (Cntrl). 3D viability); Figure 4 shows binding free energies of different monosaccharides and disaccharides studied with and without glycine molecules; FIG. 5 shows the penetration of water molecules on the amorphous cellulose surface of nanofibril cellulose with 300 mM sucrose, with 300 mM trehalose, and with 150 mM trehalose and 333 mM glycine; FIG. 6 shows SEM images of excipient-free (NFC), 300 mM sucrose (NFC+SUC), 300 mM trehalose (NFC+TRE), lyophilized nanofibrillar cellulose (NFC) formulations, and the same compositions. Enlarged image is shown.

DETAILED DESCRIPTION OF THE DISCLOSURE Methods are provided for controlled lyophilization of hydrogels comprising nanoscale cellulose to enable room temperature storage and successful reconstitution of lyophilized hydrogels that are particularly suitable for cell culture scaffolds. be done.

Surprisingly, it has been found that lyophilization and/or reconstitution of hydrogels comprising nanoscale cellulose can be controlled by adjusting the residual water content of the lyophilized hydrogel composition. For example, the use of biomolecules as cryoprotectants or lyoprotectants during lyophilization of hydrogels containing nanoscale cellulose, such as nanofibril cellulose, regulates the residual moisture content of lyophilized hydrogel compositions. , so that reconstitution of lyophilized hydrogel compositions can be improved and controlled. Typically, the biomolecule is one or more oligosaccharides, such as tri- or disaccharides, and adjustments are made by optimizing the amount of biomolecule added to the composition.

According to some embodiments of the present disclosure, the method includes adding biomolecules to the hydrogel comprising nanoscale cellulose prior to freeze-drying the hydrogel. Typically, the biomolecule is an oligosaccharide or disaccharide, preferably a disaccharide or a mixture of several disaccharides, more preferably a disaccharide, most preferably lactose (4-O-β-D-galactopyrano syl-D-glucopyranose), trehalose (α-D-glucopyranosyl-α-D-glucopyranoside) or sucrose (β-D-fructofuranosyl-α-D-glucopyranoside). Trehalose and sucrose typically both have high glass transition temperatures and hydrogen bonding capabilities. Amino acids such as glycine can be added, for example, to improve the attraction of poly- or disaccharides to cellulose. Typically, amino acids are: charged amino acids such as arginine, lysine, aspartic acid, or glutamic acid; polar amino acids, such as glutamine, asparagine, serine, threonine, tyrosine, histidine, or cysteine; parental amino acids, such as tryptophan, tyrosine, or methionine. or a hydrophobic amino acid such as glycine, alanine, valine, isoleucine, leucine, proline, phenylalanine, or methionine, preferably one of glycine, tryptophan, leucine and/or glutamine.

According to some embodiments of the present disclosure, lyophilization of the hydrogels of the present disclosure is performed at a temperature of maximum freezing, taking into account the Tg' (glass transition temperature of the maximum freeze-concentration sample) of the mixture comprising the hydrogel and biomolecules. By ensuring that the glass transition temperature (Tg) of the concentrated sample is not exceeded, it can be controlled by utilizing a lyophilization cycle.

As used herein and in the claims, the following terms have the meanings defined below.

The term nanoscale cellulose refers to nanofibrillar cellulose and/or nanocrystals. As used herein, the terms nanofibrillar cellulose, nanofibrils or nanofibrillated cellulose or NFC refer to plant-derived nanofibrillar structures comprising fibrils and fibril bundles liberated from cellulosic fibrous material or cellulose pulp. understood to include. Nanofibrillar structures liberated from cellulosic fibrous raw materials are characterized by a high aspect ratio (length/diameter): their length can exceed 1 μm, while their diameter is typically 200 nm. remains less than The smallest nanofibrils are on the scale of elementary fibrils and their diameters typically range from 2 to 12 nm. The dimensions and size distribution of fibrils depend on their disintegration method and efficiency, as well as pretreatment. Typically, the median length of the fibrils or fibril bundles in NFC is 100 μm or less, such as in the range of 1-50 μm, and the number average diameter of the fibrils or fibril bundles is 200 nm. less than, suitably in the range of 2-100 nm. Intact, non-fibrillated microfibril units may be present in nanofibril cellulose, but only in insignificant amounts. The nomenclature associated with nanofibrillar cellulose is not uniform and the literature uses inconsistent terminology. For example, the following terms have been used as synonyms for nanofibril cellulose: cellulose nanofiber, nanofibril cellulose (CNF), nanofibrillar cellulose, nanoscale fibrillated cellulose, microfibril cellulose. , cellulose microfibrils, microfibrillated cellulose (MFC), and fibril cellulose. As used herein, nanofibrillar cellulose is not meant to include non-fibrillar rod-shaped cellulose nanocrystals or whiskers. Nanocrystals are highly crystalline materials called cellulose nanocrystals (CNC), cellulose nanocrystals (NCC), or cellulose nanowhiskers (CNW). Nanocrystals are rod-like, rigid, have a narrow size distribution, and are shorter than nanofibrils. Nanocrystals also have lower viscosity and yield strength and are not as good at retaining water as nanofibrillar cellulose.

The term biomolecule, also called biological molecule, is a substance produced by cells and organisms. Biomolecules have a wide range of sizes and structures and various functions. The four major types of biomolecules are carbohydrates, lipids, nucleic acids, and proteins. As used herein, biomolecules of particular interest are carbohydrates, preferably oligosaccharides such as trisaccharides and disaccharides. The disaccharide can be a reducing sugar such as lactose, maltose and cellobiose, or a non-reducing disaccharide such as trehalose and sucrose, preferably the disaccharide is a non-reducing disaccharide.

Amino acids can also be used as biomolecules along with oligosaccharides and/or disaccharides. charged amino acids such as arginine, lysine, aspartic acid, or glutamic acid; polar amino acids, such as glutamine, asparagine, serine, threonine, tyrosine, histidine, or cysteine; amphipathic amino acids, such as tryptophan, tyrosine, or methionine; or It can be a hydrophobic amino acid such as glycine, alanine, valine, isoleucine, leucine, proline, phenylalanine, or methionine.

Typically, the freeze-drying process consists of three major steps: 1) freezing (solidification of the sample), 2) primary drying (sublimation of frozen water), and 3) secondary drying (removal of unfrozen water). divided.

The term freezing step refers to the initial step of lyophilization in which the temperature of the composition is lowered below its freezing point to solidify the sample. Freezing or general crystallization of aqueous solutions consists of two steps: 1) nucleation and 2) growth of the nuclei into macroscopic crystals. The driving force for nucleation is primarily determined by supercooling. As the temperature drops below the freezing point of the solution, the probability of forming thermodynamically desirable ice clusters that act as nuclei for ice crystallization increases.

The term primary drying refers to the main step of drying, which is usually the longest step in the freeze-drying process. During the primary drying step, frozen solid water (ice) is sublimed under reduced pressure. The driving force for sublimation is the vapor pressure difference (temperature difference) between the frozen water and the condenser surface.

The term secondary drying refers to the final part of lyophilization in which any unfrozen water still present in the sample is removed. Secondary drying can be performed at elevated temperatures because ice is removed during primary drying, thus minimizing the risk of melting or crumbling. Especially for amorphous materials the temperature rise is preferably done with a slow ramp (approximately 1° C./min) to avoid decay.

The dryness, ie the water content (or residual water content) of a freeze-dried product, is typically determined gravimetrically. The amount of water is determined by subtracting the dry weight from the initial weight. Moisture content is then calculated as the amount of water divided by dry weight or total weight, depending on the method of reporting. Another way of describing dryness is the dry substance content (DS content), also called dry matter content. DS is the percentage of solids in a mixture of substances and the unit of DS content is % by weight. The higher this ratio, the drier the mixture.

The term residual water refers to water of the freeze-dried composition that is not released during secondary drying, i.e. residual water remaining in/on the freeze-dried hydrogel composition comprising nanoscale cellulose, such as nanofibril cellulose. Residual water content of lyophilized hydrogel compositions comprising nanoscale cellulose, such as nanofibril cellulose, can be measured by Karl Fischer titration. Water that is not released from the composition during primary drying, but is removed during secondary drying is called bound water.

The term glass transition temperature (Tg') of a maximum freeze-concentration sample is the temperature at which a composition undergoes a glass transition, i.e., the temperature at which freeze-concentration of a solute has increased the viscosity of the solution until solid ice can no longer form. point to

The term reconstitution refers to the process of reconstituting a desiccant by adding a reconstitution medium, such as water and/or cell culture medium, to it. The reconstitution can be pristine or can be of different consistency or consistency, for example by adding less reconstitution medium than the released water.

According to embodiments of the present disclosure, the nanoscale cellulose is selected from nanofibril cellulose and/or nanocrystals, preferably from natural nanofibril cellulose and/or nanocrystals, more preferably the nanoscale cellulose is nanofibril cellulose , most preferably natural nanofibrillar cellulose. In hydrogels comprising nanofibrillar cellulose, the total fiber content is typically 0.5% to 5%, preferably 0.7% to 3%, more preferably 0.8% to 2.5%, most Preferably about 0.8% or 2% (m/v).

Typically, in embodiments of the present disclosure, the freeze-drying of the method includes a freezing step, primary drying and secondary drying. According to some embodiments of the present disclosure, lyophilization comprises:
After gradually lowering the temperature by 0.5-2° C./min, preferably about 1° C./min, the temperature is preferably between −40° C. and −60° C. for 1-5 hours at ATM pressure. freezing process,
The temperature is increased by 0.5-2° C./min, preferably about 1° C./min to a temperature between −40° C. and −50° C., and the pressure is increased to between 30 mTorr and 100 mTorr, preferably to a pressure of about 50 mTorr. primary drying, which is reduced and then maintained at said temperature for 2-120 hours, preferably 10-100 hours, more preferably 50-100 hours; C./min from a temperature between -40.degree. C. and -50.degree. C. to room temperature (+20.degree. C. to 25.degree. Includes secondary drying. Primary dryness can be determined by comparative pressure measurements using a capacitance manometer (absolute pressure, CM) and a pirani gauche (relative vacuum).

Typically, in embodiments of the present disclosure, hydrogel compositions are prepared by mixing different concentrations of biomolecules with water, such as ultrapure water, e.g., MQ water, which is then added to the desired total Fiber content (m/v) is combined with a hydrogel containing nanoscale cellulose, such as nanofibrillar cellulose. Alternatively, the biomolecule is added as part of the nanofibrillar cellulose manufacturing process prior to lyophilization. Preferably, the hydrogel compositions are homogenized by mixing them using conventional techniques, such as vortexing, or for more concentrated compositions using syringe techniques. The total concentration of biomolecules in the hydrogel composition is typically selected from an amount between 100 mM and 700 mM, preferably between 150 mM and 500 mM, more preferably between 150 mM and 300 mM of the total concentration of the mixture, said amount comprising: from a range between two of the following: Including being selected.

The method of the present disclosure typically comprises the steps of: a) providing a hydrogel comprising nanoscale cellulose;
b) providing at least one biomolecule selected from oligosaccharides, disaccharides, or any combination thereof;
c) mixing the hydrogel of step a) with the biomolecule of step b) to obtain a mixture; and d) lyophilizing the mixture of step c) to obtain a lyophilized hydrogel comprising nanoscale cellulose. .

By utilizing the methods of the present disclosure, hydrogel freeze-drying is controlled. A lyophilized hydrogel composition typically comprises a hydrogel comprising nanoscale cellulose and a biomolecule. It has been shown that the residual water content of a lyophilized hydrogel composition can be adjusted to control the reconstitution of the lyophilized hydrogel. Without being bound by any model of explanation, preliminary observations suggest that biomolecules penetrate nanoscale cellulose, thus increasing the surface area over which water molecules can more readily bind and/or permeate the cellulose structure. , suggesting that the addition of biomolecules to nanoscale cellulose before freeze-drying results in an exfoliation effect in addition to binding water to themselves, while placing more space on the nanoscale cellulose. do. The higher the attraction of biomolecules to amorphous NFC, the more water will pass through the system.

A lyophilized hydrogel composition is also provided, the lyophilized hydrogel composition comprising nanoscale cellulose and at least one biomolecule selected from oligosaccharides and disaccharides, and the residual water content of the lyophilized hydrogel composition comprising: 0.2% (w/w) to less than 2% (w/w), preferably 0.2% to 1.9%, more preferably 0.3% to 1.6%, most preferably 0.5 % to 1.3%. Typically, in embodiments of the present disclosure, the freeze-dried hydrogel composition comprises individual fibrous ribbons. The lyophilized hydrogel composition is typically placed on a support, preferably in or on a bag, bottle, column, syringe or well plate, more preferably glass vials, 48 well plates, depending on the intended use. , 96-well plates, 384-well plates or 1536-well plates. Storage is carried out in moisture-free, sealed packages. Depending on the intended use, storing or storing a substantially dry powder of a lyophilized hydrogel composition, i.e., aerogels according to the methods, processes and products of the present disclosure, can be compared to storing and storing wet hydrogels. provides several advantages. Aerogels become hydrogels upon rehydration. In one embodiment of the present disclosure, there are varying amounts of lyophilized nanocellulose in the wells. Adding the same amount of water or cell culture medium to each well provides a test panel with different cell culture concentrations.

To provide lyophilized hydrogel microparticles or lyophilized hydrogel droplets that can be reconstituted into microbeads; e.g., lyophilized as sheets or in molds and can be wetted, i.e., reconstituted into hydrogel scaffolds or soft membranes or to provide a lyophilized hydrogel film, i.e., an airgel film; The methods and products of the present disclosure can also be used to provide microchips. One type of hydrogel scaffold of the present disclosure is rigid reconstituted with higher solids content, typically 3%-5%, preferably 3%-4% (m/v) nanofibrillar cellulose content. membrane. Hydrogel scaffolds can be stacked, for example, to form a co-culture system with cells on top between the scaffolds. Additionally, lyophilized hydrogels can be mixed with active pharmaceutical ingredient powders (APIs) using nanocellulose as a dispersing agent when wet.

According to embodiments of the present disclosure, the residual water mole fraction is 5% to 25%, preferably 6% to 20%, more preferably 9% to 15% of the lyophilized hydrogel composition; is 50% to 90%, preferably 55% to 85%, more preferably 60% to 80% of the freeze-dried hydrogel composition; and/or the mole fraction of nanoscale cellulose is 3-40%, preferably 10%-35%, more preferably 15%-30% of the dry hydrogel composition, preferably the nanoscale cellulose is nanofibrillar cellulose.

Further provided is a lyophilized hydrogel composition obtainable by the method of the present disclosure.

Reconstitution of the lyophilized hydrogel composition includes adding a reconstitution medium to the lyophilized hydrogel composition. The reconstitution medium is typically water and/or cell culture medium or mixtures thereof. It may also include the addition of other substances or elements, for example; cells; buffers such as phosphate-buffered saline (PBS); reinforcing materials; covering materials; active agents; The volume of reconstitution medium depends on the desired hydrogel concentration. Typically, the volume corresponds to the volume of water lost during lyophilization, but can be adjusted, eg, to optimize for a particular cell type.

A preferred use of the lyophilized hydrogel compositions of the present disclosure is for cell culture. In embodiments of the present disclosure, the cells are typically prokaryotic or eukaryotic cells, preferably differentiated or undifferentiated eukaryotic cells, more preferably totipotent, pluripotent, pluripotent multipotent, oligopotent, or unipotent stem cells. Cells can be, for example, a hepatocellular carcinoma cell line (HepG2), a prostate cancer cell line (PC3), or human adipose/stromal cells (hASC). Typically the cells can be prokaryotic or eukaryotic. For example, microbial cells such as bacterial cells or yeast cells may be included. Eukaryotic cells can be plant or animal cells. The cells can be cultured cells. Examples of eukaryotic cells include transplantable cells such as stem cells, eg totipotent, pluripotent, multipotent, oligopotent or unipotent cells. In the case of human embryonic stem cells, the cells may be from deposited cell lines, or may be generated from unfertilized eggs, ie, "parthenogenetic" eggs, or from eggs that have been parthenogenetically activated. such that the human embryo is not destroyed. Cells can be maintained and grown on or in hydrogels without extracellularly generated animal or human based chemicals. Cells may be evenly dispersed on or in the hydrogel. Examples of cells thus include stem cells, undifferentiated cells, progenitor cells, and fully differentiated cells, and combinations thereof. In some examples, the cells comprise cell types selected from the group consisting of keratocytes, keratinocytes, fibroblasts, epithelial cells, and combinations thereof. In some examples, the cells are stem cells, progenitor cells, precursor cells, connective tissue cells, epithelial cells, muscle cells, nerve cells, endothelial cells, fibroblasts, keratinocytes, smooth muscle cells. cells, stromal cells, mesenchymal cells, immune system cells, hematopoietic cells, dendritic cells, hair follicle cells, platelets, red blood cells, mononuclear cells, retinal pigment cells, hepatocellular carcinoma cells and combinations thereof be done.

Additionally, a cell culture scaffold is provided, the cell culture scaffold comprising a reconstituted lyophilized hydrogel composition of the present disclosure. Typically, a cell culture scaffold further comprises water and/or cell culture medium or a combination thereof, optionally with cells.

Also provided is a method of manufacturing a reconstituted hydrogel, such as a cell culture scaffold, comprising the steps of: a) providing a lyophilized hydrogel composition of the present disclosure;
b) obtaining a reconstituted hydrogel by adding water, cell culture medium or a mixture thereof to the lyophilized hydrogel composition to obtain a reconstituted hydrogel.

According to embodiments of the present disclosure, the cell culture medium may contain cells, or cells may be added later in or on the cell culture scaffold. The method includes adding other substances or other elements in or on top of the scaffold, e.g., buffers such as NaCl or salts, or adding cell culture media and/or cells on top of the scaffold. It can contain more.

EXAMPLES The natural nanofibrillated cellulose (NFC) hydrogels used in the examples were provided by UPM, Kymmene, Finland. The concentration of NFC was 1.5% (m/v) (GrowDex™).

Analytical methods used in the examples
Rheology measurement
Viscosity, loss modulus (G″) and storage modulus (G′) measurements were performed in triplicate on selected NFC hydrogel compositions using a HAAKE Viscotester iQ Rheometer (Thermo Scientific, Karlsruhe, Germany). The instrument included a Peltier system for temperature control and all measurements were performed at 24° C. Data from rheological measurements were analyzed with HAAKE RheoWin 4.0 software (Thermo Fischer Scientific) on reconstituted lyophilized NFC hydrogels. Compositions were allowed to stand at room temperature prior to measurement.

For shear viscosity measurements, a plate-on-plate geometry with parallel 35 mm diameter steel was used for all selected compositions. The shear rate was increased from 0.1 to 1000 1/s and results were observed at 16 time points. Oscillatory frequency sweep analysis was performed in a double-cap geometry with parallel 25 mm diameter steel. A constant amplitude sweep was performed prior to the measurement to determine the linear viscoelastic region. A constant angular frequency ω=1 Hz at an oscillatory stress between 1*10−4 and 500 Pa was used. Based on the linear viscoelastic regime, the selected oscillatory stress was τ=1.5 Pa for all selected NFC hydrogel compositions and the angular frequency ranged from 0.6 to 125.7 rad s−1. Met. Results from moduli were observed at 37 time points.

Lyophilization protocol Lyophilization was performed in a laboratory-scale lyophilizer Lyostar II ((SP Scientific Inc., USA). Different lyophilization cycles were used based on the Tg′ temperature of different NFC hydrogel compositions.

Evaluation of reconstitution of lyophilized hydrogels After lyophilization, the elegance of the cake formed was evaluated and the lyophilized NFC hydrogel compositions were reconstituted with ultrapure water to the starting volume immediately after opening the cap of the vial. The speed of reconstitution of the dry composition into a hydrogel and the maintenance of composition homogeneity were evaluated. Air bubbles formed in the reconstituted hydrogel were removed by centrifugation (1000 rpm) for 1 minute.

Residual Moisture Residual moisture of the lyophilized NFC hydrogel compositions was measured with an automatic colorimetric Karl Fischer titrator (Metrohm, 899 Coulometer, Switzerland). Hydranal (Coulomat AG, Fluca) was used as the solvent for the measurements. The lyophilized NFC hydrogel composition was diluted with 1 ml of methanol and aspirated into a 0.8 ml volume syringe, which was injected into the bath flask of the Karl Fischer titrator. Results were expressed in micrograms of water and the water content of methanol was considered blank in the calculations. The weight percent and mole fraction of residual moisture in the lyophilized composition were calculated manually. All measurements were performed in triplicate.

Scanning electron microscope (SEM)
The morphology of the freeze-dried NFC hydrogel composition was studied with a scanning electron microscope Quanta FEG250 (SEM, FEI Company, USA). Sample preparation was performed manually by cutting the sample with tweezers for detection of the internal structure of the sample and surface analysis. Samples were placed on double-sided carbon tape with silver paint. Prior to imaging, samples were sputtered with platinum for 25 seconds in an Agar sputter device (Agar Scientific Ltd., UK). The homogeneity of the reconstituted lyophilized NFC-hydrogel composition was evaluated by optical microscopy (Leica microsystems, Germany).

Example 1 Evaluation of different hydrogel compositions, optimization of rheological and physicochemical properties of freeze-dried hydrogels Natural nanofibrillated cellulose (NFC) hydrogels with a fiber content of 1.5% (m/v) were grown in Finland provided by UPM, Kymmene (GrowDex™). All chemicals used in this study, D-(+)-trehalose dihydrate, sucrose and glycine (99%) were purchased from Sigma Aldrich, USA.

All studied NFC hydrogel compositions were mixed with different concentrations of trehalose, sucrose, or combinations of these biomolecules with ultrapure water (MQ water) and then added to 0.8% (m/v) total fiber Prepared by combining with NFC hydrogel up to content. The total concentration of biomolecules in the NFC hydrogel composition was between 50 mM and 1000 mM. The NFC hydrogel composition was then homogenized by vortex mixing, and a composition of biomolecules at a concentration greater than 600 mM was added to the composition by Paukkonen H. et al. ”, International journal of pharmaceuticals 532 (2017) 269-280). An NFC hydrogel with a fiber content of 0.8% (m/v) without biomolecules or organic solvents was used as a control.

The physicochemical properties of the NFC hydrogel compositions were evaluated by measuring osmotic pressure and pH. The osmotic pressure of the reconstituted lyophilized composition was measured with a manual freezing point osmometer (Osmomat 3000, Gonotech). The osmometer was calibrated to 0 mOsmol/kg with ultrapure water and from 100-850 mOsmol/kg with calibration standards (NaCl, Gonotech). For measurement, the sample was pipetted into a measurement container (Gonotech) and then placed in the upper probe of the instrument. The pH from all reconstituted lyophilized NFC hydrogel compositions was measured with pH paper (Macherey-Nagel, Germany). Measurements were repeated after lyophilization and reconstitution.

To optimize the freeze-drying temperature, the glass transition temperatures (Tg') of all NFC hydrogel compositions and controls were measured by differential scanning calorimetry (DSC) (TA instruments). The NFC hydrogel composition was pipetted into a sealed T-Zero pan (40 μl). Measurements were made by first decreasing the temperature from +40° C. to −80° C. and then heating to +20° C. at 10° C./min with a Celpurge gas flow (N2) of 50 ml/min. Temperature and heat flow were calibrated with indium. Glass transition temperature measurements were performed in triplicate and the results were analyzed with TRIOS software (TA Instruments). Two NFC hydrogel compositions containing 150 mM trehalose, 333 mM glycine and 700 mM DMSO or glycerol and having a Tg' lower than -50°C were excluded.

Freeze-drying was performed in a laboratory-scale freeze-dryer Lyostar II (SP Scientific Inc., USA). Two different lyophilization cycles were used based on the Tg' temperatures of different NFC hydrogel compositions. The freezing step was performed at −55° C./−47° C. (1° C./min) for 2 hours, after which the temperature was increased at 1° C./min to −50° C./−42° C. and the pressure was reduced to 100 mTorr/50 mTorr. . Primary drying was determined by comparative pressure measurements using a capacitance manometer (absolute pressure, CM) and Pirani Gauche (relative vacuum). Freeze-drying was then continued until secondary drying, where the temperature was gradually increased from -50°C/-47°C to +20°C at 1°C/min. The total length of the first lyophilization cycle was 54 hours and the second was 143 hours. Sample volumes were 0.2 ml or 1.75 ml depending on the intended experiment. The NFC hydrogel compositions were lyophilized in Schott Toplyo™ injection vials (Adelphi) of 2 ml or 6 ml volume, depending on the sample size. Vials were closed with rubber Daikyo D Sigma lyophilization stoppers (Adelphi). After lyophilization, All-Aluminum Crimp seals (West Pharmaceutical Sevices) were placed in vials. Freeze-dried samples were stored at +4° C. before further experiments.

The cakes of lyophilized NFC hydrogel compositions containing 150 mM trehalose and 666 mM glycine collapsed, and those containing 750 mM-1000 mM trehalose showed shrinkage. Slight cracking of the cake formed was observed in the lyophilized control and lyophilized NFC hydrogel compositions containing 50 mM to 250 mM sucrose or trehalose. An elegant, white solid cake was obtained using lyophilized NFC hydrogel compositions containing 300 mM sucrose and/or trehalose and those containing 150 mM trehalose and 333 mM glycine.

Freeze-dried (FD) NFC hydrogel compositions were reconstituted with ultrapure water (MQ water) to the starting volume immediately after opening the cap of the vial. The speed of reconstitution of the dry composition into a hydrogel and the maintenance of homogeneity of the NFC composition were evaluated.

Failure to dissolve the collapsed and shrunk cakes when the original volume of ultrapure water was added was the reason for their exclusion. Additionally, the lyophilized control formed aggregates after reconstitution which could not be homogenized.

After reconstitution of a lyophilized NFC hydrogel composition with the appearance of a successful cake, the color of the cake changed to clear immediately and a hydrogel was formed after 2 minutes of gentle manual agitation. The samples were centrifuged (1000 rpm) for 1 minute to remove air bubbles formed inside the reconstituted lyophilized NFC-hydrogel composition, after which they were transferred to the pre-lyophilized NFC-hydrogel composition. corresponding to.

Physico-chemical properties were evaluated by measuring the osmotic pressure and pH of reconstituted lyophilized NFC hydrogel compositions containing different concentrations of trehalose, sucrose, and glycine. Osmolality increased with increasing biomolecule concentration, and compositions with concentrations of trehalose or sucrose between 50 mM and 300 mM had corresponding osmolality between 50 mOsmol/kg and 300 mOsmol/kg. A slight decrease in osmotic pressure could be observed after lyophilization and reconstitution of all NFC hydrogel compositions.

Optical microscope images showed similar homogeneity between the reconstituted lyophilized NFC composition containing biomolecules and the NFC composition containing biomolecules before lyophilization. Light microscope images after the freeze-drying process showed disorganized, crystallized features, so no homogeneity could be observed from the reconstituted freeze-dried controls.

Reconstituted lyophilized NFC hydrogel compositions containing both 300 mM trehalose and sucrose had osmotic pressures higher than the calibrated range 0-850 mOsmol/kg and were excluded from further experiments. The pH of all compositions was between 6-7 and remained the same after lyophilization and reconstitution.

After optimization of the freeze-drying process, the retained properties of the hydrogel and reconstituted of the freeze-dried NFC composition had to be ensured.

Based on the organized porous structure of the cake after lyophilization, viscosity measurements were performed on three different reconstituted lyophilized NFC hydrogel compositions containing 300 mM sucrose or trehalose, and 150 mM trehalose and 333 mM glycine. I went about something containing Oscillatory frequency sweep analysis was performed on the last two compositions containing only 300 mM sucrose or trehalose based on viscosity measurements and a more appropriate Tg' temperature above -40°C.

The addition of biomolecules did not significantly affect the viscosity properties of the pre-lyophilized NFC hydrogel compositions, which decreased linearly with increasing shear rate. Unlike the reconstituted lyophilized control of FIG. 1A, the presence of biomolecules in the reconstituted lyophilized NFC hydrogel formulations preserved shear viscosity as observed from FIGS. . Compositions containing sucrose in FIG. 1A) or compositions containing a combination of glycine and trehalose in FIG. 1B exhibited equal viscosity profiles before and after lyophilization.

The viscoelastic properties of the last two reconstituted lyophilized NFC hydrogel compositions containing 300 mM sucrose or trehalose and that of the control were studied. As shown in FIGS. 2A-C, G′ before freeze-drying was equal between NFC-hydrogel compositions with and without biomolecules, which exceeded G″. After lyophilization and reconstitution, the moduli of the controls crossed each other and were significantly different from those before lyophilization, as shown in Fig. 2B. The lyophilized NFC hydrogel composition had a G' modulus equal to that before lyophilization As shown in Figure 2C, the G' of the reconstituted lyophilized NFC hydrogel composition containing 300 mM trehalose was Slightly reduced compared to the same composition before the drying process.

The weight percent residual water in the lyophilized NFC hydrogel composition was measured to ensure that the rest of the composition was lyophilized correctly. Except for the control, all compositions contained less than 3% (w/w) residual water.

To better describe the water percentage of the lyophilized material, the residual moisture mole fraction was calculated. Table 1 shows the results. The molecular weight of the NFC was taken to be that of the glucose monomers in the calculation, and the water mole fraction was theoretically calculated to be greater than 99% in all pre-lyophilized NFC hydrogel compositions. After lyophilization, the residual moisture mole fraction in the lyophilized control was determined to be approximately 35%. All freeze-dried NFC hydrogel compositions showed residual moisture mole fractions of less than 30%.

As can be seen from the results, the amount of biomolecules affects the residual moisture and the molar fraction of residual water. Therefore, it is possible to optimize the residual water content by adding different biomolecules and biomolecular weights to the hydrogel prior to its freeze-drying in order to obtain a reconstitutable lyophilized hydrogel.

Based on these results, lyophilization of hydrogels containing NFC hydrogels was particularly successful with NFC hydrogel compositions containing 300 mM sucrose as the biomolecule. Having sucrose as the sole cryoprotectant compound with NFC hydrogels has many advantages compared to other studied biomolecules. For example, the primary drying temperature could be increased from -50°C to -42°C compared to NFC hydrogel compositions containing glycine. Furthermore, the porous and organized morphology and the physico-chemical properties such as iso-osmotic solution, pH-neutral environment, and non-toxicity of NFC hydrogel compositions containing sucrose are useful for different applications. useful for

Example 2 Preparation of NFC Compositions for Cell Culture Lyophilized hydrogels were prepared using 300 mM sucrose and 1.5% nanofibril cellulose (NFC). Sucrose was mixed into NFC hydrogels and samples were lyophilized on low-adhesion 96-well plates (LyoStar II, SP Scientific).

Nanofibrillar cellulose was a natural NFC hydrogel (GrowDex™) with a fiber content of 1.5% (m/v) provided by UPM, Kymmene, Finland, and sucrose was purchased from Sigma Aldrich, USA. did.

The lyophilization cycle was as follows. Freezing was performed by decreasing the temperature at 1°C/min until a temperature of -47°C was reached. Freezing was continued in the ATM for 3 hours. Primary drying was performed at a temperature of −42° C. and a pressure of 50 mTorr (6.67 Pa) for 94 hours. During the secondary drying, the temperature was increased gradually by increasing the temperature from -42°C to 20°C at 1°C/min. The pressure was 50 mTorr (6.67 Pa) and secondary drying was continued for 1.5 hours.
The lyophilized hydrogels were packaged, sealed from atmospheric moisture and stored at room temperature for several months.

Example 3 Culture of 3D Cell Spheroids in Lyophilized and Reconstituted NFC Hydrogels The suitability of the lyophilized and reconstituted NFC hydrogels of Example 2 for 3D cell culture was studied. The cells studied were a hepatocellular carcinoma cell line (HepG2), a prostate cancer cell line (PC3), and human adipose/stromal cells (hASC).

Lyophilized 1.5% NFC hydrogel samples were directly reconstituted to different NFC concentrations. Samples were reconstituted with a mixture of MQ water, NaCl, cell culture medium and/or cells. Ph was controlled to be within the physiological range. The cell culture medium was a bicarbonate buffered nutrient mixture containing fetal bovine serum. For reconstituted lyophilized hydrogels reconstituted with MQ water only, the volume used was the volume of water lost during lyophilization. For mixtures of MQ water and cell culture medium, the amount of MQ water was the volume of water lost during lyophilization, and for cell culture medium, the amount required for dilution.

The NFC hydrogel concentration of reconstituted hydrogels containing MQ water, cell media and cells was adjusted to the optimal hydrogel stiffness for the selected cell type. The concentrations used were 0.8%, 1.0% and 0.125% for HepG2, PC3 and hASC cells respectively. The storage modulus (G′) and loss modulus (G″) of the reconstituted compositions were measured with a Viscotester (Thermo Scientific) and compared to those of the pristine hydrogels. (Gonotec) and optimized for cell culture (280-320 mOsmol/kg) by changing the composition of the reconstitution medium.The osmolarity of the reconstituted NFC hydrogel composition, It was concluded that the pH, storage modulus and loss modulus remained unchanged after freeze-drying and reconstitution.Reconstitution of native NFC to different native NFC concentrations after freeze-drying was associated with natural NFC This was possible regardless of the starting concentration.

The reconstituted NFC hydrogel, medium, and cells were mixed by pipetting and reservoir medium was added on top of the hydrogel-cell-suspension. 3D cell spheroids were prepared as described in Bhattacharya et al., “Nanofibrillar cellulose hydrogel promotes three-dimensional liver cell culture”, Journal of controlled release: official journal of the Controlled Release Society. 164 (2012) 291-8. a) Incubated at 37° C. in CO ₂ with a previously established protocol, the same protocol was used for both the reconstituted hydrogels and the non-lyophilized hydrogels used as controls. Viability of cell spheroids was examined by alamarBlue cell viability assay at 7 days, ie 1, 3, 5, 7 days. Cell spheroid size was assessed microscopically over the 7-day study period. Viability test results are shown in FIG. 3A for HepG2 cell spheroids and in FIG. 3B for PC3 cell spheroids. The left column shows the lyophilized NFC results and the right column is the control.

Studies have shown that lyophilized hydrogels can be reconstituted at different concentrations, and that reconstituted lyophilized hydrogels, like raw, non-lyophilized hydrogels, are suitable for cell culture. Both HepG2 and PC3 cells were successfully cultured on lyophilized and reconstituted native NFC hydrogels for 7 days.

Example 4 Molecular Dynamics Simulations Interactions between water, excipients, and NFC were studied with molecular dynamics (MD) simulations. MD simulations were performed to study the dynamic behavior of the system and gain insight into specific mechanisms.

Three plane-like simulation systems were developed: one with a hydrophobic surface and one with a hydrophilic surface, crystalline forms of cellulose to determine the binding free energy. , as well as amorphous forms of cellulose to determine the effect of biomolecules on water permeation.

To equilibrated water systems, excipients were added at random locations in the water phase at concentrations according to the number of water molecules. Three new systems were then generated from each system containing the following concentrations of biomolecules: 300 mM trehalose, 150 mM trehalose with 333 mM glycine, and 300 mM sucrose. Additional simulation systems were generated for different mono- and disaccharides with the following concentrations of biomolecules: 300 mM fructose, glucose, lactose, sucrose, trehalose and xylitol, 75 mM glycine and 225 mM of each saccharide combined and 200 mM of each saccharide combined with 100 mM glycine.

Three properties were of particular interest: the free energy difference between the excipient and the cellulose layer, the penetration of water into the amorphous system, and the chain-exfoliation effect of sugar in the crystalline system that was noticed during visualization. Free energy differences were determined by generating a mean force potential from the partial densities of the excipients through the gmx density. The more stable side of the cellulose plane was used to quantify the free energy difference. Water penetration was calculated using gmx select as the average number of water molecules within 1 nm of the plane along the x and y axes formed by the center of mass of the cellulose. The average number of glucose in the same region was determined in the same way, except using the carbon molecule and dividing it by 6. Finally gmx rms was used for the 8 strands on the top and bottom of both microcrystalline cellulose layers to determine the RMSD of their individual strands, which relates strand detachment as a function of time. gave the measurements.

The binding results of excipients to NFC surfaces were consistent between different cellulose surfaces. Additional simulation results with different mono- and disaccharides show that all sugar molecules prefer to concentrate at the NFC-water interface. However, the coupling strength differs between systems. As seen in Figure 4, the highest binding free energies were recorded for sucrose and lactose. Low surface interactions were found for fructose, glucose, and xylitol. All saccharides left column shows results for 300 mM saccharide, middle column shows results for 225 mM each saccharide combined with 75 mM glycine, right column shows results for 200 mM each saccharide combined with 100 mM glycine. Show the results.

Glycine was shown to have no attraction to the NFC surface. In the 300 mM concentration system, sucrose proved to have a higher attraction to cellulose surfaces than trehalose. For the hydrophobic cellulose surface, the difference between the free energies was 0.54 kJ/mol. Similarly, in a system containing half the concentration of trehalose and 333 mM glycine, the attractive force of trehalose increased slightly but significantly, with a difference between the free energies of 0.56 kJ/mol. The respective differences from the hydrophilic surface were 0.38 kJ/mol and 0.20 kJ/mol. Additional studies comparing 300 mM sucrose and 200 nM sucrose with 100 mM of one of glycine, leucine, tryptophan or glutamine showed that amino acids in general improved the attraction of sucrose to cellulose. Both tryptophan and glutamine penetrated the cellulose surface, but glutamine showed no attraction. Tryptophan showed a significantly higher attractive force than sucrose. Also, sugar-cellulose hydrogen bonding appears to increase with the addition of amino acids (except glycine).

Biomolecules had different effects on residual moisture. As shown in Figure 5, water molecules readily penetrated the amorphous cellulose surface, unlike sugar, despite their attractive force. In the absence of excipients, an average of 263 (±13 SD) water molecules were present within a 2 nm slice of amorphous cellulose. With added sucrose, water penetration increased to 274 (±15 SD). However, added trehalose had no effect (262±16 SD). A reduced amount of trehalose with glycine increased water permeation slightly to 267 (±12 SD). By determining the average number of glucose molecules in the same region, the following water:glucose ratios were obtained: 1.385 for pure water, 1.424 for 300 mM sucrose, 1.378 for 300 mM trehalose. , and 1.400 at 150 mM trehalose with 333 mM glycine. This could be explained by the different attractive forces of the biomolecules on the nanocellulose system; the higher the attractive force of the biomolecules on the amorphous NFC, the more water passed through the system. The reason for this may be that the tighter attractive force gives more space for water molecules to access the interior of the amorphous NFC. For example, the ability of water to penetrate more into amorphous NFC with sucrose than with trehalose can be advantageous in reconstituting lyophilized NFC hydrogel compositions.

During visualization, a chain-peeling effect in the crystalline system was discovered. The cellulose chains are not connected between the periodic images, so the chain ends at the junction are free to move in the water. This effect was not significant in pure water systems. However, added sugars, especially trehalose, were able to exfoliate these chain ends further into the aqueous phase. Furthermore, after pulling the chain away from the crystal structure, the sugar was able to migrate into the cleft that had formed, preventing the chain from returning. To quantify this effect, the root mean square deviation (RMSD) of the surface chains was determined as a function of time. As the chain ends are pulled from the structured crystal structure into the disordered aqueous phase, their RMSD increases. For the hydrophobic system, the cutoff for strands pulling away from the crystal structure was ˜0.15 nm and for the hydrophilic system ˜0.35 nm. Surface chains on hydrophilic surfaces are more mobile due to their hydrophilicity. The results show that trehalose and sucrose molecules can promote chain detachment on the NFC surface compared to excipient-free systems.

At higher magnification, fibrous structures about 15 nm wide stood out from the SEM image of the freeze-dried control. Individual fiber ribbons were also observable from freeze-dried compositions containing sucrose or trehalose. FIG. 6 shows an SEM image of a lyophilized NFC composition containing 300 mM sucrose and 300 mM trehalose without excipients and a magnified image of the same composition. Arrows indicate examples of individual fiber ribbons observed in the composition. This can be caused by the ability of these biomolecules to unchain, as demonstrated in crystalline NFC simulations.

In light of the present simulation results, it is clear that trehalose and sucrose molecules can interact with the NFC surface and change the structure of NFC by promoting surface chain exfoliation, which suggests that the morphology of NFC after FD This partly explains the experimentally documented differences in . Although glycine has no attraction to the NFC surface, the simulation emphasized the effect of glycine on bound trehalose on the NFC surface. Thus, in the glycine and trehalose hybrid system, the binding of trehalose to the NFC surface was slightly stronger, reaching the same binding free energy value when compared to sucrose without glycine. Furthermore, the simulation results suggested that on crystalline and hydrophobic NFC surfaces containing glycine, no chain detachment of NFC occurred, suggesting that the NFC matrix in the aqueous environment before and after freeze-drying was more retained. can provide enhanced stability and structure.

Claims (21)

A method of controlling freeze-drying of a hydrogel containing nanoscale cellulose, the method comprising adjusting the residual moisture content of the freeze-dried hydrogel. Claim 1, characterized in that the freeze-drying comprises a freezing step, primary drying and secondary drying, and the residual water is water remaining in the freeze-dried hydrogel that is not released during the secondary drying. described method. The nanoscale cellulose is selected from nanofibril cellulose and/or nanocrystals, preferably natural nanofibril cellulose and/or nanocrystals, more preferably the nanoscale cellulose is nanofibril cellulose, most preferably natural nanofibril cellulose. 3. A method according to claim 1 or 2, characterized in that The method comprises the steps of: a) providing a hydrogel comprising nanoscale cellulose;
b) providing at least one biomolecule selected from oligosaccharides or disaccharides;
c) mixing the hydrogel of step a) with the biomolecule of step b) to obtain a mixture; and d) lyophilizing the mixture of step c) to obtain a lyophilized hydrogel comprising nanoscale cellulose. A method according to any preceding claim, characterized in that 5. Process according to claim 4, characterized in that the biomolecule is a disaccharide, more preferably a non-reducing disaccharide or lactose, most preferably lactose, trehalose or sucrose. Method according to any of claims 4-5, characterized in that the amount of biomolecule is between 100mM and 700mM, preferably between 150mM and 500mM, more preferably between 150mM and 300mM of the total concentration of the mixture. The residual water content of the lyophilized hydrogel composition is between 0.2% (w/w) and less than 2% (w/w), preferably between 0.2% and 1.9%, more preferably between 0.3% and 1 6%, most preferably between 0.5% and 1.3%. A lyophilized hydrogel composition, wherein the lyophilized hydrogel composition comprises nanoscale cellulose and a biomolecule selected from oligosaccharides and disaccharides, and wherein the residual water content of the lyophilized hydrogel composition is 0.5. 2% (w/w) to less than 2% (w/w), preferably 0.2% to 1.9%, more preferably 0.3% to 1.6%, most preferably 0.5% to A lyophilized hydrogel composition, characterized in that it is 1.3%. 9. The freeze-dried hydrogel composition of claim 8, wherein said freeze-dried hydrogel composition comprises individual fiber ribbons. The composition is lyophilized on a support, preferably a bag, bottle, column, syringe or well plate, more preferably on a glass vial, 48 well plate, 96 well plate, 384 well plate or 1536 well plate. Freeze-dried hydrogel composition according to any of claims 8-9, characterized in that Claims 8-10, characterized in that the molar fraction of residual water is between 5% and 25%, preferably between 6% and 20%, more preferably between 9% and 15% of the freeze-dried hydrogel composition. The lyophilized hydrogel composition of any of Claims 8-11, characterized in that the mole fraction of biomolecules is between 50% and 90%, preferably between 55% and 85%, more preferably between 60% and 80% of the freeze-dried hydrogel composition. The lyophilized hydrogel composition of any one of Claims. The mole fraction of nanoscale cellulose is 3-40%, preferably 10%-35%, more preferably 15%-30% of the freeze-dried hydrogel composition, preferably nanoscale cellulose is nanofibril cellulose The freeze-dried hydrogel composition according to any one of claims 8 to 12, characterized in that it is A freeze-dried hydrogel composition obtainable by the method of any of claims 1-7. for cell culture; for producing hydrogel microbeads; for producing hydrated sheets or membranes; for producing prefilled microchips; or dispersing agents in mixtures with active pharmaceutical ingredients Use of the lyophilized hydrogel composition of any of claims 8-13 as a 16. Use according to claim 15, characterized in that the freeze-dried hydrogel is reconstituted with water; water and cell culture medium; cell culture medium; cell culture medium and cells; or water, cell culture medium and cells. The cell is a prokaryotic or eukaryotic cell, preferably a differentiated or undifferentiated eukaryotic cell, more preferably a totipotent, pluripotent, multipotent, oligopotent or unipotent stem cell Use according to any of claims 15-16, characterized in that A scaffold for cell culture, characterized in that it comprises a reconstituted lyophilized hydrogel composition according to any of claims 8-13. A method of manufacturing a reconstituted hydrogel, said method comprising the steps of a) providing a lyophilized hydrogel composition according to any of claims 8-13;
b) obtaining a reconstituted hydrogel by adding a reconstitution medium comprising water, cell culture medium or a mixture thereof to the lyophilized hydrogel composition to obtain a reconstituted hydrogel. ,Method. 20. Method according to claim 19, characterized in that the reconstituted hydrogel is used for cell culture. 21. A method according to claim 19 or 20, characterized in that the cell culture medium contains cells.

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