CA2213089A1 - Method of encapsulating living cellular material in an artifical matrix for culture or regeneration - Google Patents

Abstract

The present invention relates to a method to encapsulate living biologicals or biological derived materials within a three-dimensionally-shaped artificial matrix produced by freezing-thawing a Poly(vinyl alcohol) based aqueous solutions containing one aprotic co-solvent and possibly additives. Freezing-thawing of the polymeric aqueous solution around the living biological inclusions can be performed simultaneously to the storage freezing-thawing processing of biologicals between 0°C and -80°C. Resulting materials are translucent PVA-based hydrogels with living biologicals included within the hydrogel structures. Such biohybrid PVA-cells materials can be used for culturing and replacing biological tissues such as the articular cartilage.

Description

METHOD OF ENCAPSULATING LIVING c~rr-urAR MATERIAL IN AN
ARTIFICIAL MATRIX FOR CULTURE OR R~G~N~R~TION

RPCKGROUND OF THE INVENTION
(a) Field of the Invention The invention relates to a method of encapsu-lating living isolated cells, living cellular spheroids or solid inclusions within a three-dimensionally shaped polymer-based hydro-gel structure. The resulting hydro-gel-cells material can be used for in vi tro tissue or organ culture, engineering and in vivo regeneration.
(b) Description of Prior Art Formation of polymer-cells constructs has been recently promoted for culturing artificial tissues in vitro, delivering and retransplanting living cells or tissue-like biomaterials in vivo to operate tissue repair or reconstruction. Polymer-cells constructs gen-erally consist of three-dimensional polymeric matrices where living biologicals and especially cells are seeded and entrapped. Engineering and retransplantation experiments with biohybrid polymeric biomaterials-living cells constructs have specifically targeted cartilages, bone, valves, ligaments and tendons as well as metabolic organs such the liver.
Most artificial biohybrid materials have been realized on the basis of the association of engineered biopolymeric or polymeric structures with living single cells, cellular spheroids or pre-prepared solid inclu-sions. Artificial polymeric materials were selected preferably among textile constructions (non-woven, knitted or woven structures), sponges or foams as well as other dry porous polymeric constructs (porous rigid materials). A large number of biodegradable materials have been introduced as supporting matrices for biohy-brid materials including natural materials such as polypeptides (Gelatin, Collagens), polysaccharides (Hyaluronic Acid, Chitin, Chitosan) or inorganics (Coral, Hydroxyapatite), and synthetic polymers such as linear polyesters (poly[lactic acid], poly[glycolic acid], poly[lactic-co-glycolic], polylactones, polyhy-droxy-alkanoates...), poly[ethylene oxide], Poly(vinyl alcohol), polyurethanes, etc... The encapsulation of living biologicals (cells, enzymes...) within artifi-cial polymeric microbeads was rather introduced as organ equivalents for treating metabolic deficiencies, and diseased or deficient organs (liver, pancreas...).
For instance, polymers or copolymers of acrylic or methacrylic esters, polyphosphazene as well as many polypeptides (Gelatin, Poly-L-lysine...) or polysaccha-rides (Agar, Alginate, Carageenan...) were selected to encapsulate living cells within dry or gelified micro-capsules and to re-introduce them in vivo.
Poly(vinyl alcohol) (PVA) as biomaterials is a well-known water-soluble polymer since the beginning of the 1950's. Dry PVA products were evaluated in cardio-vascular or abdominal surgery (arteries, abdominalwalls) where commercially available IVALON~ PVA
sponges were intensively experimented in animals for anevrysms, pulmonary resection, mitral valves atrial wall, choledochoduodenostomy and lesions to liver and spleen. Clinical applications of IVALON~ PVA sponges were presented in 1957 for arterial replacements in 317 patients. In Orthopaedy, IVALON~ sponges were evalu-ated for arthroplasties (tibial) as soon as in 1957 and 1960, and up to 1966. However, they did not induce sufficient interest among the surgical community or provide adequate long-term per~ormances in vivo in regard to the needs expressed at this time. As a conse-quence, dry PVA products did not expand among the selected implantable medical devices.

Poly(vinyl alcohol) hydrogels (PVA-H) have recently received much interest as biomaterials, par-ticularly for drug, proteins or cells delivery systems and reconstructive devices. PVA-H products were gener-ally produced through chemical (cross-linking agents) or physical (irradiation) methods or by thermal proc-essing (freezing-thawing) of aqueous PVA solutions (Hyan S.H. et al. (1989) Polymer Bulletin, 22:119-122).
Until recently, chemical and freezing, freeze-drying or freeze-thawing techniques for processing PVA-H materials have been promoted intensively by many authors (Trieu H. et al. (1995) Polymer, 36(13):2531-2539), and associated patents: Nambu et al. (US Patent No. 4,664,857), Kobayashi et al. (US Patent No.
5,141,973). Most medical applications of PVA-H involved the drug delivery, the treatment of wound healing and the ophtalmic products. Enhancing the mechanical prop-erties, integrity and stability of PVA-H was the main goal of the various proposed methods of processing.
Poly(vinyl alcohol) derivatives with acryloyl-urethane or stilbazolium products were also gelated for wound healing or ophtalmic products. Hydrophobically-modified PVA polymers were also obtained through urethanization and treatments with acid compounds (Garba O. et al.
(1996) Polymer, 37:1183-1188) or by grafting polymeric chains of hydroxyacids such the poly(lactic acid) [PLA]
(Oka M. et al. (1996) Development of Artificial Articular Cartilage. SIROT'96 Meeting, p24, Amsterdam, The Netherlands), and may be suitable for hydrogel for-mation (Onyari J.M. et al. (1996) Polymer Reprints,145-146).
Artificial articular cartilage has been pro-posed from PVA-Hs, characterized in vitro (Peppas N.A.
(1979) Biomat. Med. Devices Artif Organs, 7(3):421-433) and evaluated in vivo for reconstructing cartilage defects in canine femoral condyles (Oka M. et al.
(1996) Development of Artificial Articular Cartilage.
SIROT'96 Meeting, p24, Amsterdam, The Netherlands). In the US 4,988,761 patent, Ikada et al. described the thermal processing of a low-water-content (<40%) PVA-H
at low and high temperatures for applying this improved PVA-H materials for human cartilage replacement. In a similar way, it was proposed that PVA-H may be used with Ceramics or Metals for artificial intervertebral disks (US 5,458,643 patent) or artificial bone connec-tion prosthesis (US 5,314,478 patent).
Microencapsulation or encapsulation of biologi-cals remains quite limited with PVA materials. Micro-capsules of PVA polymer were proposed by Solenberger et al. (US 3,941,728 patent), however there was no encap-sulation of biologicals such as enzymes or cells within PVA-H materials. Entrapment of pancreatic Islets enclosed in a mesh and within a tube-shaped PVA-H was studied in vitro and in vivo, and promoted for bioarti-ficial pancreas in treating diabetes mellitus. However,entrapment of biologicals did not correspond strictly to encapsulation or microencapsulation of biologicals where the encapsulant is continuously formed around the biologicals. Microorganisms were immobilized within a spherical hydrated gel made of about 3 to about 40% by weight of polyvinyl alcohol and about 0.2 to about 4%
of ionic polysaccharide. The ionic polysaccharide such the Alginate was cross-linked through polyvalent metal ion while the polyvinyl alcohol was gelated by at least one freezing-thawing at a temperature not higher then -5~C, preferentially at -20~C. Immobilization of enzymes and other microorganisms was performed with PVA
hydrogels by freezing-thawing as soon as in 1972. PVA
gel processing does not involve chemicals that may be toxic to cells, and PVA gels have high water content and porous structure that may help in immobilizing liv-ing microorganisms. PVA gelation into beads or spheri-cal shapes was first proposed in 1983, then improved in terms of processing parameters in 1987. PVA gelled microbeads with immobilized microorganisms were finally proposed in 1986 by boric acid and freezing-thawing cross-linking methods.
Translucent or transparent water insoluble PVA
hydrogels were cross-linked physically as described by Truter et al. (European Patent application published under No. 583,170 on February 16, 1994). PVA polymer was complexed with a chemical agent, then gelated physically by freezing. These PVA-H materials were obtained by combining PVA with a suitable aprotic sol-vent and allowing the PVA to form a semicrystallinestructure by freezing the PVA solution. After molding, the hydrogel formation takes place at -20~C in 8-17 hours. Complexing agents such the methyl vinyl ether/maleic anhydride and poly(acrylic acid) were added to the PVA solution, then frozen for hydrogel formation. PVA (8-30), Dimethyl sulfoxide (10-80) and water (20-90) permits to obtain such pure PVA-Hs.
Studies on hydrogel formation and hydrogel properties by freezing or freezing/thawing of PVA/water/aprotic co-solvent systems were proposed by many authors (Hyan S.H. et al. (1989) Polymer Bulletin, 22:119-122; Trieu H. et al. (1995) Polymer, 36(13):2531-2539). Transparent PVA-Hs were processed by dissolving 15% w/v 99,5% hydrolysis PVA polymers in 80:20 DMSO/water solutions at high temperatures (100-140~C) for hours and under nitrogen atmosphere. Casting of the PVA mixtures was performed at 60~C and PVA gela-tion was obtained at -20~C after 10 hours (Hyan S.H. et al. (1989) Polymer Bulletin, 22:119-122). For compari-son, translucent PVA-Hs were processed during 10 hours at -20~C, then at 5~C, without the presence of DMSO. In comparison to translucent PVA-Hs, transparent PVA-Hs demonstrated enhanced properties (light transmittance x 2; tensile strength x 2; elongation x 2) at same water content. In a similar way, the freezing-thawing gelation of 99,3% hydrolyzed PVA polymer in DMSO/water solution was analyzed by experimental design with the quenching temperature, the DMSO concentration and the PVA concentration as planning parameters. DMSO/water ratios were ranged from 0:100 to 75:25, PVA concentra-tions varied from 4 to 12% w/v and the quenching tem-peratures were selected at -20~C and -60~C. Highest swelling rates were obtained for low DMSO concentra-tions (25%) and high quenching temperatures (-20~C/0~C) but did not seem to change greatly with the PVA con-centration. Light transmissions were maximal for inter-mediate PVA concentrations (6-8%) and quenching tem-peratures (-20~C/-40~C) but did not vary significantly with the DMSO concentrations. Shore hardness values were maximal for high DMSO (65-75%) and PVA (10-12%) concentrations but did not change with the quenching temperature. Tensile strengths were optimal for high PVA (10-12%) and DMSO (65-75%) concentrations and low quenching temperatures (-60~C) while elongations were maximal for high PVA concentrations (8-10%) and for intermediate to high DMSO concentrations (-45~C/-60~C).
Finally, tensile resistances were superior for high PVA
(10-12%) and DMS0 (65-75%) concentrations and low quenching temperatures (-20~C/0~C). From the results obtained in the study of Trieu et al. (Trieu H. et al.
( 1995 ) Polymer, 36(13):2531-2539), PVA-Hs formed at -60~C from a 8% PVA in 50:50 DMSO/water solution will show a medium water swelling (88-89%), medium light transmission (60%), intermediate hardness (25 Shore 00), medium tensile strength (68 psi), high elongation (350-360~) and low tear resistance (3 psi).

SUMMARY OF THE INVENTION
The present invention relates to a method for encapsulating living biologicals that consist in iso-lated cells, cellular spheroids or cellular solid inclusions within thermally-processed and reversible monolithic transparent Poly(vinyl alcohol) hydrogels such that biohybrid cells-hydrogel materials are formed and available for further in vitro and in vivo manipu-lations.
In accordance with one embodiment of the pre-sent invention, the cells-hydrogel construct can be molded and consequently shaped according to complex geometry.
In accordance with one embodiment of the pre-sent invention, the method involves the steps of:
a) forming a living cellular material that con-tains the isolated cells, the culture medium supplemented with any additional dissolved com-ponents and at least one cryo-preservation pro-tective agent, and possibly the supporting artificial materials;
b) cryo-preserving the living cellular materials such that solid living cellular materials are formed and collected individually to be main-tained at low-temperature;
c) dissolving the PVA polymer under appropriate conditions in a solvent/aprotic co-solvent sys-tem so that a clear PVA solution is obtained and ready to gelate;
d) preparing the PVA solution within the desired molding materials;

e) disposing the PVA solutions within the molds at the selected gelation temperature and preparing them to gelate;
f) incorporating the solid living cellular materi-als into the PVA solutions within the molds that were maintained at the gelation tempera-ture, the solid living cellular materials being stored at normal freezing-preservation tempera-ture of animal/human cells;
g) at the first gelation signs of the PVA solu-tions, homogenizing the PVA solutions with the incorporated cellular inclusions, and maintain-ing the molds at the gelation temperature until the PVA solutions gelate and form molded PVA
hydrogels; and h) de-freezing rapidly the molds containing the cellular inclusions encapsulated within the PVA
hydrogels up to a low positive temperature, and washing repetitively the cellular inclu-sions/PVA hydrogel constructs with fresh cul-ture medium to eliminate the aprotic co-sol-vent.
Surprisingly, and in accordance with the pre-sent invention, it was discovered that the thermally driven formation of PVA hydrogel may be compatible to a certain extent with the cryo-preservation or freezing procedures of living biologicals so that biohybrid con-structs can be produced and further used for three-dimensional cultures of artificial tissues.
In accordance with another embodiment of the present invention, an aprotic co-solvent is added to the aqueous solutions to protect them from a freezing at the selected low temperatures, this co-solvent being also known and used simultaneously at low concentra-tions as a protecting agent during the cryo-storage of g living cells. Except this co-solvent, there is no other specific chemicals used to initiate or favor the cross-linkage of the PVA hydrogels of the present invention.
For the purpose of the present invention the following terms are defined below.
Herein, ~LSIs~ are defined as being solid mate-rials that are basically composed of living animal or human cells or cellular matters, one cell-compatible aqueous medium, at least one medium-compatible cryo-storage preservative agent and possibly one artificialsolid material.
~Cells~ or ~cellular matter~ is defined by a living structure, composed of a mass of protoplasm, enclosed in a membrane and containing a nucleus. Living animal or human cells may consist in metabolically ac-tive modified or unmodified cells originating from primary cells isolated from animal or human tissues, e.g. stem cells, chondrocytes, osteoblasts, tenocytes or fibroblasts, endothelial cells, keratinocytes, etc, or in established immortalized cell lines that are available from commercial sources or home-made in labo-ratories.
Cell-compatible aqueous media may consist in any standard or supplemented cell culture medium ena-bling to keep the animal or human cells metabolically active. This medium may be selected from any standard or supplemented cell culture media that are currently accepted or applied for the monolayer culture of the previously defined animal or human cells.
~Cryo-storage preservative agent~ or ~cryoprotectant~ is defined as being any water or aque-ous medium-soluble or -miscible substances that pro-tects the cells and its components during the cryo-preservation or low-temperature procedures. They may correspond to the current substances that are used for cryo-preserving and storing the animal or human cells or tissues in cell biology laboratories. Permeating cryoprotectants such as dimethyl sulfoxide or glycerol penetrate the cell membrane and reduce the intracellu-lar water concentration, and thereby limits the quan-tity of ice formed at any temperature. Nonpermeating cryprotectants such as polyvinylpyrrilidone, hydroxyethyl starch, monosaccharides or sugar alcohol operates by acting directly on the cell membranes.
Changes in the colloidal osmotic pressure and of the water-membrane interactions seem to be the main mecha-nisms for the nonpermeating protection.
Artificial solid materials may be used to carry on the animal or human cells such as to immobilize the previously defined cells onto specific materials or structures and/or according to specific density or spa-tial disposition. Animal or human cells are immobilized onto or within the artificial solid materials by inocu-lating or seeding, adhering and culturing procedures that are conducted previously to the LSIs preparation and storage. Such artificial solid materials must en-able, or at least be compatible with or not disturb, their own incorporation within the HM and must remain solids along with the LSIs preparation, storage and incorporation within the HM. These materials may con-sist in unhydrated polymeric structures with small sizes or high porosity levels, e.g. microspheres or microbeads, textiles, sponges, films, flakes, etc.
~Cryo-preserving living biologicals~ is defined by the fact that the living biologicals within the medium where they are dispersed and in presence of one cryo-preservative agent or a mixture thereof are disposed and maintained at extreme low temperatures (-140~C to -196~C). The cryo-preserving technique may vary in terms of cooling rates, cryo-preservation temperatures, and can be obtained for example through liquid nitrogen or liquid nitrogen vapors. Freezing living biologicals is defined by the fact that the living biologicals within the medium where they are dispersed and in presence of one freezing-preservative agent or a mixture thereof are disposed and maintained at low temperatures (0 to -85~C). The freezing technique may change in terms of cooling rates or freezing final temperatures, and can be performed by using ultra low temperature freezers. The freezing step (-80~C) of biologicals may be followed by a cryo-preserving step to -140~C/-196~C. Both cryo-preserving and freezing of biologicals will require preservative agents such as to avoid any occurrence of damages to the biologicals, its macro-components and sub-components.

BRIEF DBSCRIPTION OF THE DRAWINGS
Fig. 1 illustrates the Biohybrid cells-hydrogel Material (BchM) obtained through the encapsulation within a molded PVA hydrogel (HM) of Living Solid Inclusions (LSI) in accordance with one embodiment of the present invention; and Fig. 2 illustrates the potential ranges of negative/positive temperatures given for the freezing-thawing and formation of PVA hydrogels and the cryo-preserving or freezing of living biologicals in accor-dance with one embodiment of the present invention.

DE~ATr-~n DESCRIPTION OF THE INVENTION
In a first embodiment of the invention, living isolated biologicals, for example animal or human cells, are prepared in order to form Living Solid Inclusions (LSIs). In the second embodiment, these LSIs are embedded within a artificial Hydrogel Matrix (HM).

These two operations result into a living biologi-cals/artificial matrix materials, also more conven-iently called a Biohybrid cells-hydrogel Material (BchM) when living biologicals correspond to animal or human cells.
The method of the present invention is based upon a temperature-controlled gelation of an aqueous poly-meric solution around individual LSIs. It is dependent upon the fact that a temperature-driven hydrogel forma-tion can be overlapped and compatible in terms of tem-perature range with the normal cryo-preservation proc-ess of animal and human cells or any cells-based mate-rials.
Preparation and preservation of the LSIs During the freezing process, the viability of the said cells or cellular matters may be harmed by thermal shocks. Cooling just below the freezing point of the solution is a critical step for the viability of cells. For current dimethyl sulfoxide or glycerol cry-oprotectants, cooling rates ranging from 0.3 to 10~C/
minute are usually selected between 4~C and -80~C.
LSIs may be created through the cryo-preserving and/or freezing of animal or human cells in an aqueous medium whatever the animal or human cells are previ-ously adhered onto or encapsulated within artificialsolid materials, and may be preserved through cryo-pre-serving or freezing. The LSI solid state is obtained from the freezing of the water component, the biologi-cals being dispersed within the water component, or from the presence of the artificial solid materials plus the freezing of the water component, the biologi-cals being adhered to the artificial solid materials and surrounded by the water component.

As practical procedures for the formation and storage of LSIs, two typical procedures may be pre-sented:
~ Isolated animal or human cells dispersed within a standard culture medium supplemented at a low concentration with at least one cryoprotectant can be dropped into liquid nitrogen (-196~C) to form LSIs consisting basically in cells/medium/preservative agent. Resulting LSIs can then be frozen and stored at higher negative temperatures (-80~C).
~ Isolated animal or human cells dispersed within a minimal volume of standard culture medium and in presence of at least one cryoprotectant can be inoculated, adhered onto or encapsulated within artificial solid materials such as natu-ral or synthetic polymeric biomedical products, e.g. Poly(lactic acid) [PLA] or Poly(glycolic acid) [PGA] knitted meshes or PLA, PGA, Poly(lactic-co-glycolic acid) [PLGA], Polycaprolactone [PCL], Gelatin microbeads, then stored by freezing the cells-artificial solid material inclusions at a low negative tempera-ture (-80~C).
Two main reasons explain the injury to cells during freezing. First, there exist mechanical injuries to cells that are caused during the extra- and intra-cellular ice crystal formation. Second, there exist an injury by osmotic dehydration. Extracellular ice crys-tal formation is responsible for an exposition of cells to increasingly hyperosmotic environment which is due to water sequestration at the ice crystal growth. Cells shrink due to the transport of water out of the cell in response to the osmotic imbalance. This osmotic imbal-ance is caused by the increasing extra-cellular solute concentrations. ~Osmotic effects~ that are defined herein by the alteration in the osmotic strength of the suspending media caused by conversion of water to ice or ice to water may also damage cells. This conversion induces a substantial flow of water across membranes of unfrozen cells, causing volume changes during freezing and thawing.
The viability of cells that is generally defined by the ability of frozen and thawed cells to perform their normal functions is currently expressed by the ability of the cells to reproduce, metabolize, exclude vital dyes or perform other metabolic func-tions. The combination of the freezing procedure parameters and cryoprotectants may result in the fact that the conversion of freezable intracellular water into ice as well as the osmotic dehydration are reduced. It consequently helps in enhancing the viabil-ity of cells or cellular matters during freezing-tha-wing.
Formation of PVA based HMs The formation of HMs is based upon the physical cross-linking of Poly(vinyl alcohol) PVA] based solu-tions through the freezing-thawing action. HMs may con-sist in pure PVA hydrogels, in chemically grafted PVA
[cgPVA] hydrogels or in polymeric hydrogels obtained by blending the PVA with other water-soluble polymers, these additional polymers being a minor component of the resulting HMs. For examples, chemically grafting modification of PVA polymers may include acrylamides (e.g. PVA-g-N-isopropylacrylamide, etc), urea (urethanized PVA), acids (e.g. PVA-g-decanoic acid, PVA-g-stearic acid, PVA-g-docosanoic acid, etc), poly-mer (e.g. PVA-g-Poly[Lactic Acid], PVA-g-Poly[ethylene maleic anhydride], etc) while PVA based blends may include water-soluble natural or synthetic polymers, e.g. Poly(ethylene oxide), Poly(ethylene glycol), Poly-vinylpyrrilidone, Polyacrylamides, Polyacrylic or meth-acrylic acids, Polysaccharides (e.g. Chitin, Chitosan, Hyaluronic Acid, etc), Polypeptides (e.g. Gelatin, Collagen, etc), etc...
The PVA based polymer is gelated in order to encapsulate the LSIs as well as to form a three-dimen-sionally-shaped monolithic material.
~Three-dimensional shape~ refers herein to the ability of gelating the PVA based hydrogel into any customizable shape, form and geometry in the three dimensional system. This may be obtained by molding or by a phase separation during the gelation processing.
~Monolithic~ refers herein to a solid material that is bulk-formed in one-piece with its constituent elements which form a homogeneous consistent system.
Formation of PVA hydrogels via freezing-thawing may be induced with PVA polymers having high (98-99.5%), medium (90-93%) or low (88-89%) hydrolysis rates as well as high or low molecular weights. This change of the polymer characteristics may influence the chemical, physical and mechanical performances of the resulting physically cross-linked PVA hydrogel, and particularly its integrity and stability in certain environmental conditions. It must be noted that PVA
polymers with high or medium hydrolysis rates are gen-erally used for freezing-thawing cross-linking of PVA
hydrogels whatever the molecular weight. However, gel formation occurs with pure PVA polymers having differ-ent hydrolysis rates and/or characteristics as well aswith blends of PVA polymers having different hydrolysis rates and/or characteristics.
The conventional freezing-thawing formation of PVA based hydrogels for HMs is defined by the fact that the PVA based solutions are obtained by heating PVA/aprotic co-solvent/water, cgPVA/aprotic co-sol-vent/water or PVA-polymer blend/aprotic co-sol-vent/water mixtures at high positive temperatures such as the clear resulting PVA based solutions present no air bubbles and respect the initial component propor-tions, the PVA based solutions are disposed into the selected molds and quenched during several hours at a specified temperature such as PVA-based hydrogels are formed, and finally the resulting PVA based hydrogels are warmed up and extensively washed in distilled water to extract the aprotic co-solvent and obtain clean PVA-based hydrogels.
The preservation of the initial proportions in the polymer/aprotic co-solvent/water system may be eas-ily obtained during the heating and preparation of thePVA based solutions through the use of condenser to recover the co-solvent/water vapors.
The elimination of the air bubbles from the PVA
based solutions may be performed by using vacuum or ultrasonic bath to facilitate the bubble release. This operation is made easier if the PVA based solutions are kept at a warm temperature.
The gelation of the PVA-based solutions may be reached within a temperature range from the room tem-perature (S~20~C) to low negative temperatures (:~-60~C), The lower negative gelation temperature corresponds to the minimal freezing temperature for the selected co-solvent/water system. A decreasing of the quenching temperature within this temperature range (~20/-60~C) enables higher cooling rates and faster gelations of the PVA based solutions.
The processing of the PVA based HMs for encap-sulating LSIs and forming BchMs may result from the freezing-thawing technique as previously described by respecting the conditions of compatibility with the storage of LSIs.
Formation and Preparation of the BchMs Formation of BchMs results from the incorpora-tion of LSIs during the processing of the HMs byrespecting the compatibility of the temperature range (Fig. 2).
It must be noted that the pre-formed LSIs have to be stored and manipulated before gelation at suffi-ciently low negative temperatures, the higher tempera-ture limit being defined as the freezing temperature of the aqueous medium/preservative agent solution consid-ered for the LSIs and the lower temperatures being the better temperatures. For example, standard culture medium +10% dimethyl sulfoxide solutions will freeze around -15~C. LSIs may be stored at temperatures rang-ing from -80~C to -140~C which correspond to tempera-tures currently used in cell biology for cryo-preserv-ing biological materials (Fig. 2).
It must be noted that the gelation of PVA based HMs will occur at positive or negative temperatures, for example in a temperature range from +20~C to -60~C.
Freezing or freezing-thawing gelation of PVA based HMs will consider that gelation temperatures will be nega-tive, the lower temperature enabling faster cooling and gelation but the lower acceptable temperature being the temperature at which aprotic co-solvent/water systems freeze and can not gelate (Fig. 2).
The compatible temperatures zone for the hydro-gel formation and biologicals encapsulation is definedon Fig. 2 by (X). The washing zone is defined on Fig. 2 by (Y) It must be noted that the lower acceptable tem-perature for gelation of PVA based HMs is defined as being the freezing (negative) temperature of the con-sidered polymer/aprotic co-solvent/water system. This temperature limit can be easily determined for each system through experiments.
The preparation of the BchMs may be conducted after preparing the LSIs according to one of the previ-ously described manners and after preparing the PVA
based solutions as previously reported, ~ by disposing the ungelated PVA based solution within the selected mold at the specified gela-tion temperature, the specified gelation tem-perature being superior to the lower acceptable temperature as previously defined and being com-patible with the LSIs as previously described, . by maintaining the PVA based solution within the selected mold at the specified gelation tempera-ture until the occurrence of the first optical gelation signs, . by incorporating individually the LSIs and care-fully homogenizing the PVA based solution/LSIs mixture within the selected mold, ~ by keeping the PVA based solution/LSIs mixture within the selected mold at the specified gela-tion temperature until the complete gelation of the system, ~ by warming up the gelated PVA based solu-tion/LSIs mixtures, or now the BchM, to a tem-perature superior to the higher temperature limit for LSIs, this higher temperature limit being defined as the freezing temperature of the aqueous medium/preservative agent solution con-sidered for the LSIs, ~ by washing extensively the BchM with large vol-umes of pure standard aqueous medium at a tem-perature superior to the higher temperature limit for LSIs, this higher temperature limit being defined as the freezing temperature of the aqueous medium/preservative agent solution con-sidered for the LSIs in order to rapidly extract the aprotic co-solvent from the BchM.
It must be noted that both the warming up and washing steps may be temporarily eliminated and replaced by a re-lowering of the temperature in order to cryo-preserve or store the formed BchM at the initial preservation or storage temperatures of the 10 LSIs, e.g. -80~C to -140~C. However, both warming up and washing remained only postponed since later de-freezing of the BchM to temperatures superior to the higher temperature limit for LSIs, this higher tempera-ture limit being defined as the freezing temperature of the aqueous medium/preservative agent solution consid-ered for the LSIs, or to positive temperatures will necessarily imply the warming up and washing procedures as previously described.
It must be noted that the aprotic co-solvent or preservative agent/medium and/or aprotic co-solvent or preservative agent/water mixtures may be responsible of temperature-mediated toxic activities toward living biologicals (LSIs or cells, etc), particularly at higher positive temperatures. To avoid such potential toxic reactions, the warming up and washing will be kept at temperatures that are superior to the higher temperature limit for LSIs, this higher temperature limit being defined as the freezing temperature of the aqueous medium/preservative agent solution considered for the LSIs, but inferior to the temperature at which the aprotic co-solvent or preservative agent induces toxic activities toward living biologicals. This upper temperature limit for induced toxicity is generally well defined for the preservative agents selected and used for cryo-preserving or freezing living biologi-cals.
Once the aprotic co-solvent or preservative agent is mainly eliminated from the BchM or remains temporarily at low concentrations within the BchM, the BchM may be transferred for culture procedures or any other biological uses in vitro at a temperature which is superior to the upper temperature limit for induced toxicity as previously defined, for example at a normal incubation temperature of 37~C.
Encapsulation of LSIs containing or not one artificial solid materials, e.g. microbeads, flakes, spheroids, etc and based upon living cells from normal or pathological tissues or established cell lines, e.g.
chondrogenic, osteogenic, fibroblastic, stem cells, neuronal, hepatocytic, etc, within PVA based HMs as proposed in the present invention may be applied to cellular or tissular engineering experiments for pro-ducing organ- and/or tissue-like materials. Biomedical applications of such BchM based products include engi-neering of artificial tissues and/or organs, cellular engineering as well as clinical repairing or replacing of non-functional tissues and substituting deficient metabolic functions.
The present invention will be more readily un-derstood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

EXAMPLE I
Encapsulation of cryo-preserved cellular microdisper-sions within an artificial PVA matrix Cultured living chondrogenic cells were col-lected, dispersed in a standard MEM medium (a-MEM, Minimal Essential Medium, + 15% FCS, Fetal Calf Serum, + antibiotics), then dimethyl sulfoxide [DMSO] was added at 4~C to the cells in medium dispersion to have a 10:90 (v/v) DMSO/MEM ratio The dispersion was in-jected manually by a precision micrometric pipet as small diameter microdropplets into sufficient vol-ume/surface area container with liquid nitrogen. Liquiddispersion dropplets solidified rapidly at -140~C into small size microspheres and were gently stirred to avoid any aggregation or fusion. The liquid nitrogen volume was reduced, then the liquid nitrogen excess was eliminated by evaporation at -80~C resulting in dry in-dividual (cells + DMSO + medium~ LSIs. The LSIs were maintained frozen and stored at -80~C. A PVA solution was prepared by dissolving 10% w/v PVA (99% hydrolyzed, Mw 89,000-98,000) in pure DMSO at 90-140~C for 2 hours under stirring, then the 10% w/v PVA solution was cooled down to room temperature and diluted with ster-ile standard a-MEM medium to reach final 5% w/v PVA and 50:50 DMSO:MEM proportions. The required amount of the 5% w/v PVA in 50:50 DMSO:MEM solution was transferred into the mold, then cooled down to -60~C in a low tem-perature freezer. The LSIs that still are preserved at -80~C were rapidly transferred and incorporated at -60~
C in PVA/DMSO/MEM mixture at the first optical signs of gelation, homogenized and maintained at -60~C until the Z5 complete gelation occurs (transparent to translucent system). After the gelation of the LSIs/ PVA/DMSO/MEM
mixture, the system is rapidly defrost to 0-4~C and washed abundantly with MEM medium which is renewed periodically to extract and eliminate the DMSO. The resulting BchM is a PVA-H with living chondrogenic cells included into small size chambers within the hydrogel and is prepared for cultures at 37~C (Fig. 1).
In Fig. 1, the resulting BchMs (A, B & C) have cylindrical shapes (small diameter Petri dishes as molds), but the first one (A) presents LSIs by cryo-freezing a cells-medium dispersion into microdropplets (Chambers), the second one (B) has encapsulated LSIs made of frozen medium and cells adhered onto Gelatin microspheres (Inclusions) and the third one (C) is obtained by encapsulating a frozen aqueous medium +
cells adhered onto a methyl cellulose fabrics within the PVA hydrogel (Composite).

EXAMPLE II
~nc~rsulation of cells-microspheres within an artifi-cial PVA matrix - Cho~drogenic cells adhered onto Gela-tin microsphere Living chondrogenic cells were previously inoculated and cultured onto Gelatin microspheres (120-150 ~m). The adhered cells and Gelatin microsphereswere collected individually, then rinsed in 10:90 DMSO/standard medium (a-MEM, Minimal Essential Medium, + 15% FCS, Fetal Calf Serum, + antibiotics) for some minutes. The DMSO/medium excess was eliminated and the cells + microspheres (LSIs) were frozen at -80~C in an ultra-low temperature freezer. A PVA/DMSO/medium solu-tion was prepared from 99% hydrolyzed PVA (Mw 89,000-98,000) and prepared for gelation as described in Exam-ple I. The required amount of the 5% w/v PVA in 50:50 DMSO:MEM solution was transferred into the selected mold, then cooled down to -60~C in a low temperature freezer. The LSIs that still are preserved at -80~C
were rapidly transferred and incorporated at -60~C in PVA/DMS0/MEM mixture at the first optical signs of gelation, homogenized and maintained at -60~C until the complete gelation occurs (transparent to translucent system). After the gelation of the LSIs/PVA/ DMSO/MLM
mixture, the system is rapidly defrost to 0-4~C and washed abundantly with MEM medium which is renewed periodically to extract and eliminate the DMS0. The resulting BchM is a PVA-H with living chondrogenic cells included into small-size chambers within the hydrogel and is prepared for cultures at 37~C. A
similar experiments can be performed with different materials and constructions.
While the invention has been described in con-nection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any varia-tions, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims (18)

1. A method of encapsulating living cellular material in a monolithic Poly(vinyl alcohol) (PVA) hydrogel, which comprises the steps of:
a) cryo-preserving living cellular materials to be maintained at low-temperature; and b) physical gelation of a Poly(vinyl alcohol) -based aqueous solution at negative temperatures of about -80°C to about 0°C around cryo-preserved living cellular materials of step a) to form said PVA hydrogel.

2. The method of claim 1, which further comprises a step prior to step a) which consists in:
forming living cellular material that contains isolated cells in a culture medium supplemented with at least one cryo-preservation protective agent.

3. The method of claim 2, which further comprises a step between steps a) and b) which consists in:
dissolving a PVA polymer under appropriate conditions in a solvent/aprotic co-solvent system so that a Poly(vinyl alcohol)-based aqueous solution is obtained and ready to gelate.

4. The method of claim 1, wherein said Poly(vinyl alcohol)-based aqueous solution is clear.

5. The method of claim 4, wherein step b) is effected by incorporating the living cellular materials into Poly(vinyl alcohol)-based aqueous solution within molds that were maintained at a gelation temperature.

6. The method of claim 5, which further comprises a step of homogenizing Poly(vinyl alcohol)-based aqueous solution with the incoporated living cellular materials at the first gelation signs of Poly(vinyl alcohol)-based aqueous solution.

7. The method of claim 6, which further comprises a step of defrosting the molds and washing PVA hydrogels with culture medium to remove aprotic co-solvent.

8. The method of claim 1, wherein said Poly(vinyl alcohol) solution contains at least one standard or supplemented biological aqueous medium and one aprotic co-solvent.

9. The method of claim 1, wherein the monolithic PVA hydrogel is three-dimensionally gelated and shaped in artificial molds of plastic, metal or glass materials.

10. The method of claim 3, wherein the aprotic co-solvent is dimethylsulfoxide and the cryo-preservative agent is dimethylsulfoxide.

11. The method of claim 1, wherein the living biological material is selected from the group consisting of cells, cellular matters, enzymes or bacteria of plant, animal or human origin.

12. The method of claim 11, wherein the culture medium is one that enables the culture of the said cells, cellular matters, enzymes or bacteria.

13. The method of claim 1, wherein the PVA hydrogel further comprises water-soluble substances, water-miscible substances or water-insoluble solid products.

14. The method of claim 1, wherein the PVA hydrogel further comprises solid films, microspheres, textiles or sponges.

15. The method of claim 1, wherein the Poly(vinyl alcohol) is pure high-hydrolysis level Poly(vinyl alcohol) (>93%), pure low-hydrolysis level (<93%) or of a mixture thereof.

16. The method of claim 15, wherein the Poly(vinyl alcohol) is blended or mixed with low-concentrations (<50%) water/co-solvent-soluble natural, artificial or synthetic polymers.

17. The method of claim 15, wherein the PVA further comprises organic and/or inorganic additives to enhance the properties of the resulting PVA hydrogel.

18. The method of claim 11, wherein the Poly(vinyl wherein the living biological material is selected from cartilage, ligament, tendon, bone and marrow tissues.