Substrate and method

Abstract

A substrate comprises a carrier (10) that promotes a cultivation of biological entities (30), particularly live cells, more particularly live induced pluripotent stem cells (iPSCs). Said carrier comprises an electrostatically charged exposed surface expressing a first polarity. Said surface is covered by an electrostatically charged first sacrificial layer (11) of a second polarity, opposite said first polarity. Said first sacrificial layer (11) is covered by an electrostatically charged second sacrificial layer (12) of said first polarity, wherein said second sacrificial layer comprises moieties to interact with said biologic entities (30). The entities (30) are detached from said substrate (10,11,12) by disintegrating one or more of said sacrificial layers (11,12) by means of an electrically charged agent, particularly by introducing electrically charged molecules or ions.

Description

[0001] Substrate and method

[0002] The present invention relates to a substrate, comprising a carrier that promotes a cultivation of biological entities, particularly live cells, more particularly induced pluripotent stem cells. The invention further relates to a method of cultivating biological entities, particularly live biological cells, more particularly live induced pluripotent stem cells (iPSCs).

[0003] In this respect cultivation is to be understood as comprising one or more of selectively or non-selectively capturing, tethering, maintaining, culturing, stimulation, proliferation, purification and / or harvesting of biological entities. Said biological entities may comprise biologies and organisms, particularly live cells, cytokines, or other bio-active compounds, more particularly live induced pluripotent stem cells (iPSC).

[0004] The present invention particularly relates to a substrate and harvesting procedure for capturing and colonisation of biological entities, including cells and compounds such as micro-organisms, cells, cytokines, or other bio-active molecules, serving many applications, including biological compound purification, culturing, expansion, and modification. The present invention may especially be used for the cultivation of live induced Pluripotent Stem Cells (iPSCs).

[0005] Induced pluripotent stem cells are similar to natural pluripotent stem cells, such as embryonic stem cells, in many aspects, such as the expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling time, embryoid body formation, teratoma formation, viable chimera formation, and potency and differentiability. iPSCs are generated from somatic cells that have been reprogrammed by the ectopic expression of defined embryonic transcription factors. The cells are derived from skin or blood cells that have been reprogrammed back into an embryonic-like pluripotent state that enables the development of an unlimited source of any type of human cell needed for therapeutic purposes. This technology has provided scientists with a powerful tool for modelling disease and developing treatments for human disorders. Accordingly, iPSCs are a great promise for a wide variety of applications, like the development of cell therapies, screening of new drugs, and modelling of pathologies.

[0006] Notwithstanding the above promises, challenges in mass production of iPSCs remain a key hurdle to a clinical translation. An important factor is formed by the substrate that is used for cultivation of new cells. Such substrate may comprise one or more carriers in the form of a nutritious layer, for instance kept in a Petri dish, providing a two-dimensional cultivation space, or formed by individual bodies (micro-carriers) that may be colonised all over and cultured in suspension, offering a further scalable three dimensional cultivation environment as a result of a better surface to volume ratio compared to a conventional two dimensional Petri dish culture. Although these suspension based systems seem a great promise as they potentially offer many more cell capturing anchors that could potentially boost production, these systems often tend to be expensive and ineffective due to a poor cell adhesion (often less than 80%). Moreover, these known suspension based systems prove to be relatively inefficient for the cultivation and harvesting of iPSCs due to an observed loss of their pluripotency.

[0007] It is, inter alia, an object of the present invention to provide for a cultivation substrate and associated cultivation method for biological entities that counteracts the above drawbacks while maintaining the advantages associated with an adhesion based biological purification or culture system.

[0008] To that end, a substrate according to the invention is characterized in that said carrier comprises an electrostatically charged exposed surface expressing a first polarity, wherein said surface is covered by an electrostatically charged first sacrificial layer of a second polarity, opposite said first polarity, wherein said first sacrificial layer is covered by an electrostatically charged second sacrificial layer of said first polarity, and wherein said second sacrificial layer comprises moieties to interact with said biologic entities. The first sacrificial layer, that covers the outer carrier surface, mediates an electrostatic interaction between said charged surface of said carrier and the second sacrificial layer. The second sacrificial layer offers a tunable and on-demand releasable coating that promotes the cultivation of said microbiological entities that counteracts the above drawbacks while maintaining the advantages associated with existing adherend culture systems.

[0009] This system, according to the invention, may be expanded beyond two layers of sacrificial material by repeating said polarity-driven layer construction. Particularly, said substrate may be coated with more than two layers of materials having alternating (i.e., opposite) polarities charges, resulting in a multi-layered structure with n layers, wherein at least the outermost layer is a sacrificial layer promoting tethering of biological compounds. It is further envisage that said substrate may carry only one sacrificial layer of a material having a charge with opposite polarity to that of said carrier surface, wherein said single sacrificial layer promotes tethering of biological compounds.

[0010] In view of a favourable cultivation surface to volume ration, a preferred embodiment of the substrate according to the invention is characterized in that said carrier comprises a collection of micro-carrier bodies, each comprising an electrostatically charged exposed surface expressing said first polarity and being covered by said electrostatically charged first sacrificial layer of said second and said electrostatically charged second sacrificial layer of said first polarity, respectively. This embodiment is particularly used in a suspension based cultivation system for biological entities, particularly live cells and more particularly live iPSCs.

[0011] A further specific embodiment of the substrate according to the invention is characterized in that at least part of said micro-carrier bodies are capable of interacting with biological cells, particularly by being functionalized with one or more compounds selected from a group, comprising: nucleic acids (aptamers), proteins and peptides.

[0012] Said micro-carrier bodies may serve as biological cell carriers or cell-adhesive micro-particles. These micro-carrier bodies may contain a positively charged surface for example resulting from the presence of a polyelectrolyte such as poly-l-lysine. Alternatively the micro-carrier bodies may contain a cell-adhesive (natural) polymer or a protein such as gelatin, collagen, fibronectin or laminin, or alternatively a polymer functionalized with cell-adhesive moieties such as cellular integrin binding peptide sequences containing arginine-glycine-aspartic acid (RGD) or cellular cadherin binding peptide sequences containing histidine-alanine-va line acid (HAV), or combinations of any of the above.

[0013] A further specific embodiment, the substrate according to the invention is characterized in that said micro-carrier bodies have a size in a range of between 1 micron and 5 millimetre, specifically in a range of between 1 micron and 500 micron. Particularly these micro-carrier bodies have a coefficient of variation in size of less than 10%, preferably less than 5%. In case of non-spherical micro-carrier bodies the above size refers to their Feret diameter. Micro-carrier bodies with a size between 10 and 100 micron are particularly suitable for pharmaceutical applications. For the purpose of cell culture, food and nutrition, gel micro-carrier bodies having a size between 100 and 500 micron may be used. In cosmetics, personal care and agriculture the micro-carrier bodies will generally have a size between 500 and 5000 micron. However, it will be appreciated that in any of these fields of application the particle size may also be chosen outside those ranges to suit a specific application in particular.

[0014] The final product may be a medicinal product, wherein said micro-carrier bodies comprise a pharmaceutically active agent. These micro-carrier bodies typically may have a size between 10 and 100 micron. The product may be a beauty product, wherein said micro-carrier bodies comprise a cosmetically active agent. Such particle typically may have a size between 500 and 5000 micron. The final product may be a food product, wherein said micro-carrier bodies comprise a nutritious ingredient or supplement. The micro-carrier bodies in such case may typically have a size between 100 and 500 micron.

[0015] In a specific embodiment, the substrate according to the invention is characterized in that said micro-carrier bodies are suitable for administration by intramuscular, intravenous, subcutaneous, intra-articular or intra-peritoneal injection. In a further specific embodiment, the substrate according to the invention is characterized in that said micro-carrier bodies are suitable for administration to the eye, nose, oral cavity, gastrointestinal tract or vaginal cavity.

[0016] Existing adhesion based culture systems, both on Petri dish and suspension based culture on micro-carriers, often use proteolytic enzymes such as Trypsin to detach adherent cells from the culture substrate. Trypsin is a globular pancreatic protease which cleaves at the C-terminal side of lysine and arginine, breaking down vessel-adhering proteins and allowing easy resuspension during cell harvesting, but also non-specifically stripping off proteins from the cells during harvesting, thereby causing damage to the cell and / or their extracellular matrix. Alternatively, dissolvable micro-carriers have been developed, which are known to not-fully dissolve, which effectively produces cell-sized micro-particles that contaminate the purity of the cell harvest, which challenges its use for clinical translation and associated commercial use.

[0017] The invention is based on the recognition that the electrostatic interaction between said surface and said sacrificial layers may be broken relatively easily and gently, for instance using electrically charged agents like charged molecules and ions, particularly ionic liquids, that cause disintegration of one or more of the sacrificial layers. Accordingly, a method of cultivating biological entities, particularly live biological cells, more particularly live induced pluripotent stem cells (iPSCs), is characterized in that said biological entities are cultivated on a substrate according to the invention, and in that said entities are detached from said substrate by disintegrating one or more sacrificial layer by means of an electrically charged agent, particularly by introducing electrically charged molecules or ions.

[0018] Disintegrating and / or removing a sacrificial layer will detach that layer and any sacrificial layer covering that layer from the carrier (bodies), thereby also releasing the biological entities that were tethered to the outermost sacrificial layer. It has been found that such an electro-chemically induced release causes no significant damage to said biological entities, in contrast to existing methods that are based on enzymatic degradation of proteins, for example using Trypsin. As a result, for example cells like induced pluripotent stem cells that, cultured on and harvested from such substrate, retain their viability and function, and especially their pluripotency.

[0019] In a further embodiment, the method according to the invention is characterized in that at least one sacrificial layer is disintegrated by means of an agent, comprising a cyto-compatible or bio-compatible ionic liquid, particularly an ionic liquid selected from the group of choline-based ionic liquids, amino-acid-based ionic liquids, and choline-amino acid ionic liquids, more particularly comprising choline lactate or choline acetate. Said ionic liquids may comprise one or more components from the group of ammonium, imidazolium, sulfonium, piperidinium, and pyridinium ions. Said ionic liquids may also comprise anionic bases selected from the group of halides, tetrafluoroborate, nitrate, sulfonate, carboxylate, phosphate, and amino acids. Particularly favourable results were achieved with a preferred embodiment of the method according to the invention characterized in that at least one of said sacrificial layers is disintegrated by means of an agent, comprising choline lactate or choline acetate.

[0020] Particular beneficial results may be obtained with a preferred embodiment of the substrate according to the invention, said micro-carrier bodies comprise micro-particles, particularly gel particles, having a core that comprises a polymer network expressing said first polarity. Such relatively soft micro-carrier bodies appear to support an efficient and effective bio-reactor culture, particularly for iPSCs. These soft, cell-adhesive micro-carrier bodies may be produced in monodisperse ultra-high throughput manner by combining soft bio-materials with In-air micro-fluidics technology that will be described in more detail below.

[0021] A further specific embodiment of the substrate according to the invention is characterized in that said gel particles comprise a hydrogel, more specifically a hydrophilic polymer network, and more particularly in that said hydrogel comprises one or more polysaccharides selected from agar, alginate, chitosan, dextran, polyethylene glycol), collagen, gelatine, hyaluronic acid, carrageenan, fibroin, fibronectin, poly-l-lysine ( PLL), cellulose, graphene, polyethylenimine (PEI), poly(amidoamine) (PAA), dextran sulfate, silk, silk fibroin, pectin, K-carrageenan, lota carrageenan, gellan gum, guar gum, tragacanth gum, xanthan gum, acacia gum, karaya gum, locust bean gum, or sodium carboxymethyl cellulose (S-CMC). All of these materials are preferably applied as naturally derived materials and / or synthetically derived materials including recombinant proteins and / or derivatives of these materials, wherein said polymer network particularly comprises a calcium-alginate network.

[0022] Particularly successful results were obtained in this respect with a further specific embodiment of the substrate according to the invention, wherein said gel particles comprises a cross-linked or inter-penetrating alginate network, particularly a calcium cross-linked alginate network. In this respect a particular embodiment of the substrate according to the invention is characterized in that said bio-polymer network comprises a negatively charged alginate network.

[0023] Preferably a sacrificial layer is employed that readily dissolves or otherwise disintegrates when subjected to an appropriate chemical or physical environment. To that end, a further preferred embodiment of the substrate according to the invention is characterized in that said first sacrificial layer comprises a polyelectrolyte or polysaccharide. More particularly, a further embodiment of the substrate according to the invention is characterized in that said polysaccharide comprises chitosan, particularly vegan chitosan. Chitosan is a positively charged polysaccharide that may be used beneficially within the scope of the present invention as a sacrificial intermediate between a negatively charged surface of a micro-carrier body and a negatively charged second sacrificial layer. The sacrificially coated and electro-statically charged substrate, notably comprising a collection of micro-carrier bodies, may serve as a platform and micro-biological cell reactor. In this respect, a particular embodiment of the substrate according to the invention is characterized in that said second sacrificial layer comprises a bio-polymer network expressing said second polarity and comprising one or more peptides that interact with one or more biological entities such as one or more cell surface receptors. Like any other of the sacrificial layer(s), said second sacrificial layer may be released relatively gently if exposed to an agent containing appropriately charged molecules and / or ions.

[0024] In this respect, a particular embodiment of the substrate according to the invention is characterized in that said one or more peptides comprise Arginylglycylaspartic acid (RGD). Arginylglycylaspartic acid (RGD) is known as a peptide motif responsible for cell adhesion to the extracellular matrix (ECM) found in many species, including humans. Cell adhesion proteins recognize and bind to this sequence. Depending on the application and the integrin targeted, RGD can be chemically modified or replaced by a similar peptide which promotes particular cell adhesion, notably of induced Pluripotent Stem Cells (iPSCs). A specific embodiment, accordingly, of said substrate according to the invention is characterized in that said one or more peptides are a motif for adhesion and / or stimulation of induced pluripotent stem cells (iPSCs).

[0025] A variety of bio-polymers may be used to serve as matrix within the second sacrificial layer. A specific embodiment of the substrate according to the invention, however, is characterized in that said bio-polymer network comprises a negatively charged alginate network.

[0026] Hereinafter, the invention will be described in further detail with reference to a specific, nonlimiting embodiment and an accompanying drawing. In the drawing:

[0027] Figure 1 a setup for vreating mono-disperse micro-carriers for use in a substrate according to the invention;

[0028] Figure 2 a photographic image of micro-carriers obtained with the setup of figure 1;

[0029] Figure 3 a size distribution among the micro-carriers depicted in figure 2;

[0030] Figure 4 a schematic representation of a substrate micro-body of a substrate according to the invention; Figure 5 a chart showing the proliferation of live biological cells on a substrate according to the invention;

[0031] Figure 6 a chart showing the colonization of micro-carriers by biological cells within a substrate according to the invention; and

[0032] Figure 7 a schematic representation of a method of harvesting biological cells from a substrate according to the invention.

[0033] It is noted that some figures may be drawn purely schematically and not necessarily to a same scale. In particular, certain dimensions may have been exaggerated to a more or lesser extent to aid the clarity of any features. Similar parts are generally indicated by a same reference numeral throughout the figures.

[0034] Example:

[0035] Micro-carriers 10 with a desirable 3D shape, mechano-instructive properties, and bio-adhesive surfaces, are prepared with a blended solution (1:1) of alginate (80-120 cP) and PEG-diamine (Mn ~l,500) for physical and subsequent chemical cross-linking. To generate gel microparticles, a piezo-actuated microjet 1 releasing of 1% (w / v) a interrupted jet of micro-droplets of said blended solution is allowed to collide with a continuous microjet 2 of 0.01 M CaCI2 and 10% (v / v) ethanol (EtOH). This creates a collection of micro-carriers as depicted in figure 2 consisting of calcium stabilized aliginate. As shown in figure 3 this method is capable of creating micro carrier-bodies that are mono-disperse within a narrow band. Within the scope of the present invention, these micro-bodies may have a size ranging from a few micrometre to a few millimetre, like the micro-bodies that are shown, having a size around 220 micrometre.

[0036] The aliginate particle 10 express an electrostatically charged surface having a negative polarity. Using a layer-by-layer technique, said surface is covered by a positively charged first sacrificial layer 11, as shown in figure 4, comprising chitosan, preferably vegan chitosan. Said positively charged first sacrificial layer is coated by a negatively charged second sacrificial layer 12 comprising aliginate. The latter layer is biofunctionalized by the electrostatic interaction between microgels and poly-electrolytes functionalized with RGD-peptide moieties. This technology proves to be a viable technology for the reliable ultra-high throughout generation of mono-disperse soft micro-carriers with excellent surface bio-modification capacity. After three days of culture, numerous cells had adhered to the surface of micro-carriers. Nine days post-seeding, many of the micro-carriers were linked together by cells, and numerous tridimensional structures were created by interaction between adherent cells. Figures 5 and 6 show experimental results obtained with the substrate described hereinbefore.

[0037] Enzyme-free cell detachment of cells from the micro-carriers using bio-ionic liquids is demonstrated in figure 7. Using a bionic liquid, notably choline acetate, the first and second sacrificial layers 11,12 are disintegrated, thereby releasing the biological cells 30 that were cultured on the second sacrificial layer 12. This allows for a superior, efficient, and cell-friendly cell harvesting as compared to traditional methods based on enzymatic cleavage. Particularly iPSCs may be cultivated and harvested efficiently while preserving their pluripotency.

[0038] The present invention thereby offers a promising technology for ultra-high throughput production of soft mono-disperse micro-carriers with adjustable mechanical properties in a manner that allows accurate control over the chemical structure of surface components for efficient cell expansion.

[0039] Although the invention was explained in more detail above on the basis of merely a single example and embodiment, it should be clear that the invention is by no means limited to that. On the contrary, many variations and embodiments are still possible within the scope of the invention for an average person skilled in the art.

[0040] As far as any products, compounds, compositions, formulations, devices, methods, or uses are disclosed and described in this application, it is to be understood that certain aspects are not limited to specific products, compounds, compositions, formulations, devices, methods, or uses, as these may vary without departing from the scope and spirit of the invention.

[0041] Particularly, said outer sacrificial layer may comprise a bio-active peptide sequence or peptide epitope that can interact with a cell surface receptor, cytokine, and / or other bio-active moiety, more particularly a peptide sequence that may offer binding or adhesion of cells, growth factors, extracellular matrix components, and / or ions, even more particularly a peptide sequence derived from one or more compounds selected from the group of Fibronectin, Laminin, Collagen, Nidogen, CAM, VCAM, Netrin, Thrombospondin, Cadherin, BMP-2, TGF-B1, BMP-7, VEGF, Osteonectin, Osteopontin, Angiopoeitin, FGF-2, Phage-display, BDNF, NGF, NCAM, Myxinidin, Melittin, Ovalbumin, T-cells, Enolase, H1N1, Heparin, Polysaccharide, Decorin, Amelogenin, Dentin phosphoprotein, and Statherin, more particularly one or more peptide sequences selected from the list of RGDS, GRGDS, YRGDSCGRGDS, CGRGDSY, RGDSPASSKP, KQAGDV, PRARI, IKVAV, SIKVAV, CQAASIKVAV, CSRARKQAASIKVAVSADR, YIGSR, CDPGYIGSR, CGGRKRLQ.VQLSIRT, PDSGR, RKRLQVQLSIRT, VFDNFVLK, RNIAEIIKDI, RYVVLPR, AGQWHRVSVRWG, IKLLI, SINNNR, LRGDN, POG, DGEA, GFOGER, FPGERGVEGPGP, GFPGER, NYYSNS, FRGDGQ, QIDS, LDT, DELPQLVTLPHPNLH-GPEILDVPST, QWRDTWARRLRKFQ- REKKGKCRKA, CSVTCG, HAVDI, RRETAWA, DWIVA, KIPKASSVPTELSAISTLYL, SSVPT, SDVGWNDWIV, LIANAK, KPSSAPTQLN, KLTWQELYQLKYKGI, GHK, SWYGLR, QHREDG, YRSRKYSSWYVALKR, KLPGWSG, FAQRVPP, QHLPRDH, KLPGWSG, RGIDKRHWNSQ, CTDIKGKCTGACDGK-QC, EVYVVAENQQGKSKA, WGIRRILKYGKRS, GIGAVLKVLTTGLPAL- ISWIKRKRQQ, KWKLFKKIGIGAVLKVLT, TGLPALISC, NAVSIQKKK, MAXI, MARG1, FFECG, ISQAVHAAHAEIN EAGR, SIINFEKL, KFEGTEDAV-ETIIQAIEA, SSLENFRAY, YPTFGEHKQEKDLEY,HYR, AHARDGVFDYYT, SLYNTVATL, FLIVIGSIIGPGGDGPGGD, HSNGLPL, TSPHVPYGGGS, YPVHST, WAGEGD, LRKKLGKA, LRELHLNNN, VPGIGK, YN, YS, YL,VL, FF, R, NPYHPTIPQSVH, MLPHHGA, QQRFEWEFEQQ, LEAWPATDKTKREEVD, TKREEVD, DSS, and DDDEEKFLRRIGRFG, and combinations of two or more of the above sequences offering a synergistic and additive effect such as, but not limited to, improved cell adhesion.

[0042] Such bio-active moiety or peptide sequence may be covalently or otherwise bound to said outer sacrificial layer. The bio-active moiety of peptide sequence may be modified with a moiety, such as, but not limited to a compound selected from the group of acetyl, formyl, pyro-glutamyl, fatty acid, urea, biotin, avidin, amine, amide, esters, carboxylic acid, aldehyde, thiols, Fmoc, amino acids, proteins, and enzymes.

[0043] It is also noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an active agent" includes mixtures of two or more such agents, and the like.

[0044] The present invention offers a substrate having a granular composition, the grains of which are formed by micro-carrier bodies, especially gel particles, particularly gel micro-particles. The expressions "bodies" and "particles", as used herein, are interchangeable with"spheres", "beads", "pearls" or "capsules" and, particularly refers to particles that comprise a core which is surrounded by a shielding shell and / or a core confining polymer network of a hydroplilic gel compound or composition.

[0045] The gel particles may contain an active agent or other substance dispersed, dissolved or otherwise distributed therein. The gel particles are usually made up of substantially uniform particles of a spherical shape, although the gel particles may also be irregularly shaped. The uniform gel particles can be in the size range (diameter) from submicron to millimetre. Particularly these particles have a coefficient of variation in size of less than 10%, preferably less than 5%. In case of non-spherical particles the above size refers to their Feret diameter.

[0046] The term "active agent" as used herein is interchangeable with "bio-active agent", "cosmetically active agent", "pharmaceutically active agent", or "drug" and refers to an agent which has biological activity and used to treat, diagnose, cure, mitigate, prevent (i.e., prophylactically), ameliorate, modulate, or have an otherwise favorable effect on a disease, disorder, infection, and the like. Active agents also include a pro-drug which becomes bioactive or more bioactive after it has been placed in a predetermined physiological environment.

[0047] Various forms of the active agent can be used, which are capable of being released from the gel particle into adjacent tissues or fluids. To that end, a liquid or solid active agent can be incorporated into the cores of the gel particles described herein. As such, the active agents can be acidic, basic, or amphoteric salts. In some embodiment, the active agents can be nonionic molecules, polar molecules, or molecular complexes capable of hydrogen bonding. The active agent can be included in the gel particles in the form of, for example, an uncharged molecule, a molecular complex, a salt, an ether, an ester, an amide, polymer drug conjugate, or other form to provide the effective biological or physiological activity.

[0048] Examples of active agents that can be incorporated into gel particles herein include, but are not limited to, biologicals, anti-oxidants, vitamins, hormones, vaccines, microbiotics, probiotics, prebiotics, antibiotics, enzymes, proteins, fungi, yeast, bacteria, plant cells, nucleic acids, mammalian cells and stem cells. In some embodiment, the active agent may be water-soluble or water-dispersible. In some embodiment, the active agent may be soluble or dispersible in a solvent such as organic solvent or inorganic solvent. The active agent may be dissolved or dispersed in an aqueous solvent. One non-limiting example of the aqueous solvent is water.

Claims

Claims:

1. Substrate, comprising a carrier that promotes a cultivation of biological entities, particularly live cells, more particularly live induced pluripotent stem cells (iPSC), characterized in that said carrier comprises an electrostatically charged exposed surface expressing a first polarity, wherein said surface is covered by an electrostatically charged first sacrificial layer of a second polarity, opposite said first polarity, wherein said first sacrificial layer is covered by an electrostatically charged second sacrificial layer of said first polarity, and wherein said second sacrificial layer comprises moieties to interact with said biologic entities.

2. Substrate according to claims 1, characterized in that said carrier comprises a collection of micro-carrier bodies, each comprising an electrostatically charged exposed surface expressing said first polarity and being covered by said electrostatically charged first sacrificial layer of said second and said electrostatically charged second sacrificial layer of said first polarity, respectively.

3. Substrate according to claim 1 or 2, wherein said micro-carrier bodies comprise microparticles, particularly gel particles, having a core that comprises a polymer network expressing said first polarity.

4. Substrate according to claim 3, wherein said polymer network comprises a negatively charged alginate.

5. Substrate according to anyone of the preceding claims, wherein said first sacrificial layer comprises a polyelectrolyte or polysaccharide.

6. Substrate according to claim 5, wherein said polysaccharide comprises chitosan, particularly vegan chitosan.

7. Substrate according to anyone of the preceding claims, wherein said second sacrificial layer comprises a bio-polymer network expressing said second polarity and comprising one or more peptides that interact with one or more biological entities such as one or more cell surface receptors.

8. Substrate according to claim 7 , wherein said one or more peptides comprise Arginylglycylaspartic acid (RGD).

9. Substrate according to claim 7 or 8, wherein said one or more peptides are a motif for adhesion and / or stimulation of induced pluripotent stem cells (iPSC).

10. Substrate according to anyone of claims 7 to 9, wherein said bio-polymer network comprises a negatively charged alginate network.

11. Method of cultivating of biological entities, particularly live biological cells, more particularly live induced pluripotent stem cells (iPSCs), comprising the step of cultivating biological entities on a substrate according to anyone of the preceding claims, wherein said entities are detached from said substrate by disintegrating one or more of said sacrificial layers by means of an electrically charged agent, particularly by introducing electrically charged molecules or ions.

12. Method according to claim 11, wherein at least one of said sacrificial layers is disintegrated by means of an agent, comprising a cyto-compatible or bio-compatible ionic liquid, particularly an ionic liquid selected from the group of choline-based ionic liquids, amino-acid-based ionic liquids, and choline-amino acid ionic liquids, more particularly comprising choline lactate or choline acetate.

Images