METHOD OF CULTURING ANIMAL CELLS AND ENGINEERING TISSUE AND TISSUE-LIKE STRUCTURES

Abstract

Volvox-derived beads, a method for the production thereof, and their uses as a scaffold for animal cell culture, in a method of culturing animal cells and in a method of engineering tissue and tissue-like structures. Also, compositions and pharmaceutical compositions including engineered tissue and tissue-like structures and to the cosmetic and therapeutic uses thereof.

Claims

1-15. (canceled)

16. A composition, or solid support, or scaffold for animal cell culture, comprising Volvox-derived beads, said Volvox-derived beads consisting of inactivated Volvox colonies.

17. A method of culturing animal cells comprising culturing the animal cells in a culture medium comprising a composition, or solid support, or scaffold for animal cell culture, comprising Volvox-derived beads according to claim 16, wherein the animal cells adhere to the Volvox-derived beads and proliferate around said Volvox-derived beads, thereby forming animal cell aggregates comprising animal cells and at least one Volvox-derived bead.

18. The method according to claim 17, wherein said method comprises: first contacting the animal cells to be cultured with the composition, or solid support, or scaffold for animal cell culture, comprising Volvox-derived beads, and incubating them in culture medium; then transferring the animal cells and the composition, or solid support, or scaffold for animal cell culture, comprising Volvox-derived beads, to a culture vessel and culturing the animal cells in the presence of the composition, or solid support, or scaffold for animal cell culture, comprising Volvox-derived beads.

19. The method according to claim 17, wherein the animal cells to be cultured and the Volvox-derived beads are contacted in a ratio of about 1.10.sup.6 animal cells for about 0.2 cm.sup.3 to about 4 cm.sup.3 Volvox-derived beads.

20. The method according to claim 17, wherein the animal cells to be cultured are human cells.

21. The method according to claim 17, wherein the animal cells to be cultured are human primary cells.

22. A free animal cell aggregate comprising animal cells and at least one Volvox-derived bead, said Volvox-derived bead consisting of an inactivated Volvox colony.

23. A method of engineering tissue or a tissue-like structure, comprising: culturing animal cells in a culture medium comprising a composition, or solid support, or a scaffold for animal cell culture, comprising Volvox-derived beads according to the method of claim 17, thereby obtaining free animal cell aggregates comprising animal cells and at least one Volvox-derived bead, said Volvox-derived bead consisting of an inactivated Volvox colony; transferring and culturing said animal cell aggregates on culture inserts for tissue or tissue-like engineering and culturing said animal cell aggregates, thereby obtaining tissue or a tissue-like structure.

24. A composition comprising tissue or a tissue-like structure engineered from the free animal cell aggregate of claim 22.

25. A pharmaceutical composition comprising tissue or a tissue-like structure engineered from the free animal cell aggregate of claim 22, and at least one pharmaceutically acceptable excipient.

26. A method of filling tissue in a subject in need thereof, comprising injecting to the subject the composition according to claim 24.

27. The method according to claim 26, wherein said method is for a cosmetic treatment.

28. The method according to claim 26, wherein said method is for the treatment of tissue loss or injury.

29. The method according to claim 28, wherein the tissue loss or injury is a post-surgical and/or a post-traumatic tissue loss or injury.

30. A method of producing Volvox-derived beads, comprising: culturing Volvox algae in order to obtain Volvox colonies; isolating the Volvox colonies; and inactivating the Volvox colonies, thereby producing Volvox-derived beads consisting of inactivated Volvox colonies.

31. The method according to claim 30, wherein the Volvox colonies are inactivated in ethanol at a concentration expressed in volume/volume percent (v/v) ranging from about 60% to about 80%, for at least about 1 h, at about 4° C.

32. The method according to claim 31, wherein the Volvox colonies are inactivated in ethanol at a concentration expressed in volume/volume percent (v/v) ranging from about 65% to about 75%.

33. The method according to claim 31, wherein the Volvox colonies are inactivated in ethanol for at least about 2 h.

34. The method according to claim 30, further comprising dehydrating the Volvox-derived beads, thereby obtaining dehydrated Volvox-derived beads.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0415] FIG. 1 is a group of photographs obtained with a Leica DMI1 inverted microscope equipped with a Leica MC170HD camera showing Volvox colonies. FIG. 1A is a photograph of living Volvox colonies and FIG. 1B is a photograph of Volvox colonies inactivated following a 24 h incubation in ethanol, bar: 100 μm.

[0416] FIG. 2 is a graph showing the viability assessed with a MTS cytotoxicity assay of L929 mouse fibroblasts following incubation with medium (negative control), a pure Volvox extract (100%), or toxic extracts (positive control) either pure (100%) or diluted (50%). Cell viability was expressed as a percentage of the number of viable cells after incubation in the extract considered with respect to the number of viable cells after incubation in medium (negative control).

[0417] FIG. 3 is a group of photographs showing VCBs before (FIG. 3A) and after (FIGS. 3B, C) seeding with L929 mouse fibroblasts. The photographs of FIGS. 3A and B were obtained with a Leica DMI1 inverted microscope equipped with a Leica MC170HD camera, bar 100 μm. White arrows show the L929 mouse fibroblasts attached at the surface of a VCB after 2 h of incubation (FIG. 3B). The photograph of FIG. 3C was obtained with an environmental scanning electron microscope (ESEM) and show the aggregation of mouse fibroblasts at the surface of a VCB, bar 10 μm.

[0418] FIG. 4 is a is a group of photographs showing human dermal fibroblasts seeded either on alginate beads (FIG. 4A) or on VCBs consisting of inactivated Volvox colonies (FIG. 4B). The floating aggregates of human dermal fibroblasts and VCBs were transferred on culture inserts for further incubation (FIG. 4C), leading to the engineering of a tissue-like structure (FIG. 4D). A cross-section of the tissue-like structure with a thickness up to 300 μm was observed after detection of Volvox-beads through a chemical coupling with rhodamine and of fibroblast nuclei labelled with DAPI (FIG. 4E) and after detection with hematoxylin eosin saffron staining (FIG. 4F). Bars as indicated. Photograph were obtained with a Leica DMI1 inverted microscope equipped with a Leica MC170HD camera (FIGS. 4A and B), a canon IXUS 175 camera (FIGS. 4C and D), a Leica DMI600 fluorescence microscope equipped with a DFC300FX camera (FIG. 4E) and a Leica DM1000 optical microscope equipped with a DMC 4500 camera (FIG. 4F).

[0419] FIG. 5 is a is a group of photographs showing the tissue augmentation immediately (FIG. 5A) or one month (FIG. 5B) after subcutaneous injection to a nude mouse of VCB-engineered tissue-like structures. The photographs of FIGS. 5A and B were obtained with a Canon IXUS 175 camera, black arrows indicate the tissue augmentation resulting from the injection. Tissue analysis was carried out on a biopsy performed one month after injection of bovine collagen gel (FIG. 5C) or VCB-engineered tissue-like structures (FIG. 5D). The photographs of FIGS. 5C and D were obtained with a Leica DMS1000 macroscope, bar 500 μm. Histological analysis with hematoxylin eosin saffron staining was carried out on the biopsy specimen (FIGS. 5E, F, G, H). The photographs of FIGS. 5E, F, G and H were obtained with an inverted optical microscope, bar as indicated. Black arrow indicates the presence of blood vessels (FIG. 5H).

EXAMPLES

[0420] The present invention is further illustrated by the following examples.

Example 1

[0421] Materials and Methods

[0422] Material

[0423] Volvox carteri strain (NIES-397) was obtained from the Microbial Culture Collection of the National Institute for Environmental Studies in Japan. The algae were grown in VT medium at 22° C. with a day/night cycle of 12 h/12 h and lighting of 13000 lux and 37 W/m.sup.2. VT medium was prepared according to Provasoli et al., 1959 and comprises 500 μmol/L Ca(NO.sub.3).sub.2; 235 μmol/L Na.sub.2-βglycerophosphate; 162 μmol/L MgSO.sub.4; 670 μmol/L KCl; 0.07 nmol/L vitamin B12; 0.41 nmol/L biotin; 30 nmol/L thiamine; 3.8 mmol/L glycylglycine; 8 μmol/L Na.sub.2EDTA.2H.sub.2O; 2.2 μmon FeCl.sub.3; 0.55 μmon MnCl2; 0.11 μmon ZnSO.sub.4; 0.05 μmon CoCl.sub.2; 0.036 μmol/L Na.sub.2MoO.sub.4; pH is buffered at 7.5.

[0424] L929 mice fibroblasts (ATCC, reference ATCC® CCL-1™) and neonatal human dermal fibroblasts (HDFn, Thermo Fisher Scientific, catalog number C-004-5C) were grown in DMEM medium supplemented with 10% FCS (Hyclone), 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin, thereafter referred to as complete culture medium, at 37° C. under humidified atmosphere (5% CO.sub.2, 95% air).

[0425] Alginate beads were produced from an alginate solution (MANUCOL® LKX FMC Biopolymer 1.5% in 0.9% NaCl solution) by an extrusion method using an air-jet droplet generator system. The beads were gelled in a CaCl.sub.2 bath (120 mM in 0.9% NaCl solution, pH 7.6). Cross-linked beads were washed with PBS and stored at 4° C.

[0426] Unless otherwise specified, all products were obtained from Gibco.

[0427] Methods

[0428] Preparation of Volvox-Derived Carrier Beads (VCBs)

[0429] The Volvox colonies were sieved on a stainless-steel screen with a porosity of 100 μm, then washed with PBS and centrifuged at 300 g for 5 minutes at room temperature. Volvox-derived beads, hereafter referred to as Volvox-derived carrier beads (VCBs), were obtained by incubation of Volvox colonies in 70% ethanol for at least 2 hours at 4° C.

[0430] Culture of L929 Cells with VCBs

[0431] A pellet of about 1 cm.sup.3 of VCBs was mixed with 1.10.sup.6 L929 cells in 1 mL of complete culture medium in a sterile tube. The mixture was incubated at 37° C. for 45 minutes before being transferred in a polystyrene Petri dish untreated for cell culture and further incubated for 24 h at 37° C. under humidified atmosphere (5% CO.sub.2, 95% air). Images of the culture were taken using an inverted optical microscope. The cells were rinsed in PBS, fixed in 3% glutaraldehyde in Rembaum buffer (pH 7.4) for 1 h and observed with an environmental scanning electron microscope (ESEM).

[0432] Culture of HDFn Cells with VCBs

[0433] A pellet of about 1 cm3 of VCBs was mixed with 1.10.sup.6 HDFn cells in 1 mL of complete culture medium in a 15-mL centrifuge tube. The mixture was incubated at 37° C. for 45 minutes before being transferred to a polystyrene Petri dish untreated for cell culture and further incubated for 24 h. The cells were then transferred into a 23-mm diameter polycarbonate culture insert (Nunc). 2 mL of complete culture medium were added to the upper compartment and 1 mL was added in the lower compartment. The cells were grown at 37° C. under humidified atmosphere (5% CO.sub.2, 95% air). The media was changed every 2 days for 6 days, then the upper compartment was emptied. Cells were fed with 1 mL of culture medium in the lower compartment. This medium was renewed every 2 days for two additional weeks.

[0434] Implantation of Tissue-Like Structures in Athymic Mice

[0435] All grafting experiments were done according to an animal protocol approved by the local ethic comity for animal use. A tissue-like structure engineered from VBCs seeded with human dermal fibroblasts was collected and injected subcutaneously in the back of an athymic mouse through a sterile syringe. The animal was sacrificed after one month and the skin at implantation point was harvested and observed under a macroscope. The biopsy was afterwards fixed for 1 h in 4% formalin buffer, dehydrated in a series of graded alcohols, and included in paraffin. Blocks were sectioned at 8 μm thickness using a microtome and slides were stained using a standard hematoxylin and eosin staining protocol.

[0436] Cytokine Secretion

[0437] Cytokine secretion was measured from culture supernatants at different times following the seeding of VCBs with cells. Human interleukin 6 (IL6) ELISA ready-set-go (eBiosciences) was used according to the manufacturer's instructions. Standard curves were plotted simultaneously from serial dilutions of recombinant human IL6 included in the kit.

[0438] Toxicity Test

[0439] A pure Volvox extract (100%) was obtained by incubating a pellet of about 1 cm3 of fixed Volvox colonies in 1 mL of DMEM at 37° C. under agitation for 24 h. Both a negative control, i.e., a non-toxic extract corresponding to medium only, and a positive control, i.e., a toxic extract, were obtained in parallel. DMEM (6 mL/cm.sup.2) was added both to Thermanox coverslips (non-toxic extract) and to the wells of a plate which bottom was covered with a self-curing luting composite (toxic extract).

[0440] The extracts were then deposited on monolayers of L929 fibroblasts seeded the day before in the wells of 96-well plates. The culture was extended by 24 h at 37° C. in the presence of 5% CO.sub.2.

[0441] A MTS cytotoxicity assay was carried out to assess the toxicity of the extracts. The assay is based on the reduction of MTS tetrazolium compound by viable cells to generate a colored formazan product that is soluble in cell culture media. This conversion is thought to be carried out by NAD(P)H-dependent dehydrogenase enzymes in metabolically active cells. The formazan dye produced by viable cells can be quantified by measuring the absorbance at 490-500 nm. A decrease in the absorbance at 490-500 nm reflects a decrease in the metabolic activity correlated to a decrease in the number of living cells. Following incubation with the extracts, 20 μL, of MTS reagent were thus dispensed in each well and incubated for 2 h at 37° C. The absorbance at 490 nm was measured and the cell viability was determined. Cell viability was expressed as a percentage of the number of viable cells after incubation in the extract considered with respect to the number of viable cells after incubation in the non-toxic extract (negative control). In accordance with the ISO10993 standards, an extract associated with a cell viability higher than 70% was considered as non-toxic.

[0442] Results

[0443] Volvox-Derived Carrier Beads

[0444] Volvox algae were cultivated using standard techniques and their viability was monitored through their capacity to roll and proliferate in the culture conditions (see FIG. 1A). Volvox colonies were collected by filtration and the medium was replaced with 70% ethanol, thus killing all the algae cells of the Volvox colonies (see FIG. 1B and FIG. 3A). After 12 hours in ethanol, the Volvox colonies lost their ability to float in the culture medium. No rolling could be detected and dead colonies settled at the bottom of the culture dish under the action of gravity. Further attempts to revivify the Volvox colonies through an incubation in fresh culture medium were unsuccessful, demonstrating that no living cells were present after ethanol treatment. The obtained entities were named Volvox-derived beads or Volvox-derived carrier beads (VCBs). During the incubation in ethanol, the Volvox colonies lost most of their chlorophyll pigments, which diffused into the ethanol. Some pigments were still present in the daughter colonies embedded in the mucus core of the VCBs. These pigments were found to be non-toxic for all the cell types tested. Moreover, these internal pigmented dots proved to be useful as a rapid detection means of the mucus core of VCBs when mammalian cells were cultured in the presence of VCBs.

[0445] Volvox-Derived Carrier Beads as a Support for Mammalian Fibroblast Culture

[0446] In order to test the capacity of the VCBs to serve as a cell-colonizable substrate, L929 fibroblasts, i.e., a murine cell line originating from subcutaneous tissue, were cultured at their contact (see FIG. 3). These cells are the cells of choice for cytotoxicity standardized tests such as the ISO 10993 tests. The L929 fibroblasts rapidly adhered to the globular structures of the VCBs. Indeed, after 2 hours, some fibroblasts could be seen to have attached to the outside part of the VCBs (FIG. 3B). The colonization of the VCBs continued until they were completely covered with fibroblasts. The morphology of the attached fibroblasts was examined using an environmental scanning electron microscope (ESEM). They formed small aggregates (FIG. 3C) of two or three cells tightly attached to the surface of the VCBs without any respect to the former cellular niche abridging the original algal cells. No fibroblasts could be seen penetrating the inner compartment of the VCBs.

[0447] In conclusion, the VCBs were able to efficiently promote cell attachment and proliferation at their surface, thus leading to the formation of cell aggregates.

[0448] The suitability of the VCBs as an animal cell culture support was assessed by measuring the viability percentage of the fibroblasts after their contact with Volvox extracts in a MTS cytotoxicity assay (FIG. 2). The viability of the fibroblasts after incubation with a pure Volvox extract was compared to that of the fibroblasts after incubation with a control toxic extract either pure (100%) or diluted (50%). The results of the MTS tests showed a viability of about 95% (94.2%±9.3) after incubation with the pure Volvox extract, while incubation with a pure toxic extract (100%) resulted in a complete loss of viability and incubation with a diluted toxic extract (50%) resulted in a viability of about 50% (48.3%±1.7). A viability of about 95% is markedly greater than the accepted threshold of 70% as defined according to the ISO 10993 standards, thus establishing that the pure Volvox extract is not toxic to the cells.

[0449] In conclusion, the inactivated Volvox colonies, i.e., the Volvox-derived carrier beads, make a suitable support for animal cell culture with no observed adverse effects on the viability of said animal cells.

[0450] Volvox-Derived Carrier Beads as a Support for the Production of Tissue-Like Structures

[0451] The capacity of VCBs to support the growth of human dermal cells was then tested (see FIG. 4). For this purpose, neonatal dermal fibroblasts from human foreskin (HDFn) were seeded both on alginate beads made of alginic acid, a polymer purified from brown algae, and on VCBs obtained following inactivation of Volvox colonies as described hereinabove.

[0452] A comparison between the number of human fibroblasts observed at the surface of alginate beads and that observed at the surface of VCBs demonstrated the outstanding ability of VCBs to enable cell adhesion and growth at their surface (see FIGS. 4A and B). Indeed, while HDFn cells adhered to each other without interacting in any way with the alginate beads (FIG. 4A), HDFn cells adhered rapidly to the VCBs and aggregates of groups of HDFn cells and VCBs could be observed (FIG. 4B).

[0453] In conclusion, the inactivated Volvox colonies, i.e., the Volvox-derived carrier beads, proved to be a better support for the growth and proliferation of animal cells than the commonly used alginate beads.

[0454] The floating aggregates made of groups of HDFn cells and VCBs were then transferred and cultured into culture inserts used for tissue engineering (FIG. 4C), allowing to continue this aggregation for at least three weeks. A cohesive, tissue-like structure, presenting a jelly texture, was thus obtained (FIG. 4D).

[0455] After 21 days of growth in vitro, the cultures presented a tissue-like organization (FIG. 4E) with a width varying between 50 to 300 microns depending on the quantity of HDFn cells-VCBs added in the culture wells. The VCBs could be detected through a chemical coupling with rhodamine Staining with hematoxylin and eosin demonstrated the presence of HDFn cells throughout the whole structure (FIG. 4F). No shrinkage of the tissue-like structures could be observed, as is usually observed with classical collagen-based tissues. The presence of neo-synthesized collagen was assessed by a Hematoxylin-eosin-saffron trichromatic staining in these blocks. Inside the three-dimensional structures, the fibroblasts appeared flat growing between large blocks of VCBs (FIGS. 4E and 4F). No apoptotic cells could be detected in these tissues. Finally, a measurement of the pro-inflammatory interleukin 6 (IL-6) released in the culture medium was done every two to three days for a total of two weeks of culture. During this time, no IL-6 secretion could be detected, reinforcing the non-toxicity of the VCBs and demonstrating their role as a passive but effective support to grow and carry animal cells.

[0456] In conclusion, the inactivated Volvox colonies, i.e., the Volvox-derived carrier beads, allows the engineering of healthy tissue-like structures which do not show any sign of inflammation or apoptosis.

[0457] In Vivo Transplantation of Tissue-Like Structures Engineered with Volvox-Derived Carrier Beads

[0458] In order to test their in vivo behavior, three-dimensional tissue-like structures of human dermal fibroblasts obtained as described hereinabove were subcutaneously implanted in athymic mice.

[0459] To this aim, the tissues were collected in a syringe and a total volume of 150 mm.sup.3 of jelly-like substance was injected directly under the mouse skin creating a protruding implant visible from the outside (FIG. 5A). The volume augmentation created by the injection remained detectable and unchanged in size and shape during the totality of the experiment (FIG. 5B). As a control, an equal amount of bovine collagen gel was similarly injected to mice in order to create a similar soft tissue augmentation.

[0460] After one month, at necropsy, histological analysis of biopsy specimens taken from mice implanted with the VCBs-engineered tissues showed the presence of a large mesenchymal tissue-like structure penetrated by surrounding blood vessels (FIG. 5D). By contrast, histological analysis of biopsy specimens taken from mice injected with an equal amount of bovine collagen gel showed a smaller mass that was hardly detectable under the mouse skin (FIG. 5C). Histological analyses of the biopsy specimens taken from mice implanted with the VCBs-engineered tissues also demonstrated that the implanted tissue-like structures and surrounding tissues showed no signs of inflammation, as shown by the absence of eosinophils, neutrophils and macrophages or any other inflammatory cells (FIGS. 5E, F and G). Moreover, the histological analyses showed a progressive colonization of the interstitial spaces by stromal ingrowths associated with the beginning of a vascularization of the area one month after grafting (FIG. 5H arrow).

[0461] In conclusion, the inactivated Volvox colonies, i.e., the Volvox-derived carrier beads, allows the engineering of tissue-like structures which are suitable for in vivo implantation. Indeed, implantation of said VCB engineered tissue-like structures does not induce any inflammation of the surrounding tissues. Moreover, after implantation, the VCB engineered tissue-like structures remain stable, with no sign of rapid degradation or collapse, and actually show sign of being well-integrated, as indicated by the observation of the beginning of a vascularization.