PROCESS FOR PRODUCING CARRIER PARTICLES FOR THE CULTIVATION OF BIOLOGICAL CELLS, CARRIER PARTICLES AND THEIR USE

Abstract

The invention relates to a process for producing carrier particles for the cultivation of biological cells, comprising the steps of providing an aqueous suspension of hydrogel beads and freeze-drying of the hydrogel beads so that dry hydrogel particles are formed, wherein at least one lyoprotectant substance is added to the suspension, the hydrogel beads are loaded in the suspension with the lyoprotectant substance and the dry hydrogel particles receive a shape under the effect of the lyoprotectant substance, which shape approximates a spherical particle shape and is still maintained after rehydrating. The invention also relates to carrier particles for a cultivation of biological cells, which comprise dry hydrogel particles, preferably having a protein coating, and to applications of the carrier particles.

Claims

1. A method for producing carrier particles for culturing biological cells, comprising the steps of: providing an aqueous suspension of hydrogel beads, and freeze-drying the hydrogel beads such that dried hydrogel particles are formed, wherein at least one lyoprotectant substance is added to the aqueous suspension, wherein the hydrogel beads in the aqueous suspension are loaded with the at least one lyoprotectant substance and, under the effect of the at least one lyoprotectant substance, the dried hydrogel particles obtain a shape approximated to a spherical particle shape.

2. The method according to claim 1, wherein the hydrogel beads are loaded with a protein layer.

3. The method according to claim 1, wherein residues of the at least one lyoprotectant substance are removed after the freeze-drying.

4. The method according to claim 3, wherein the residues are removed by mechanical processing of the dried particles, comprising disintegration and screening.

5. The method according to claim 1, wherein the at least one lyoprotectant substance comprises at least one of trehalose, dimethyl sulfoxide, sucrose and a poloxamer.

6. The method according to claim 1, wherein a concentration of the at least one lyoprotectant substance in the aqueous suspension is selected to be in a range from 1 mg/ml to 500 mg/ml.

7. The method according to claim 1, wherein the dried hydrogel particles are subjected to rehydration, and residues of dried lyoprotectant substance are removed before using the carrier particles in a washing solution comprising sodium chloride.

8. The method according to claim 1, wherein the hydrogel beads contain at least one of magnetic particles and biologically active substances, wherein the at least one of the magnetic particles and the biologically active substances are dried with the hydrogel beads during the freeze-drying.

9. The method according to claim 8, wherein the hydrogel beads contain differentiation factors.

10. The method according to claim 1, wherein at least one cohesion-reducing substance, which promotes pourability of the dried hydrogel particles, is added to the aqueous supension.

11. The method according to claim 10, wherein the at least one cohesion-reducing substance comprises polyethylene glycol.

12. The method according to claim 1, wherein the freeze-drying of the hydrogel beads comprises the following phases: a freezing phase, in which the hydrogel beads are frozen according to a time-temperature function with a freezing interval of at least 120 min and an end temperature of less than -40° C., a stabilization phase, in which the hydrogel beads are stored at the end temperature for a stabilization interval duration of at least 90 min, a first drying phase for forming the dried hydrogel particles, in which a first negative pressure, which is selected in a range from 30 .Math.bar to 60 .Math.bar, is applied to the frozen hydrogel beads at the end temperature, a second drying phase, in which a second negative pressure, which is lower than the first negative pressure, is applied to the dried hydrogel particles at a temperature equal to or greater than the end temperature, and a ventilation phase, in which the dried hydrogel particles are transferred to a normal pressure according to a time-pressure function having a pressure increase interval of at least 0.5 min.

13. Carrier particles for culturing biological cells, comprising dried hydrogel particles having a protein coating, wherein the dried hydrogel particles contain a lyoprotectant substance and have a shape approximated to a spherical shape.

14. The carrier particles according to claim 13, wherein the protein coating comprises at least one of Matrigel, collagen, laminin and vitronectin.

15. The carrier particles according to claim 13, wherein surfaces of the carrier particles are free of residues of the lyoprotectant substance.

16. The carrier particles according to claim 13, having a characteristic cross-sectional dimension in a range from 50 .Math.m to 2 mm.

17. The carrier particles according to claim 13, containing at least one of magnetic particles, and biologically active substances.

18. A method of using the carrier particles according to claim 13 as carrier particles for culturing biological cells.

Description

[0040] Further details and advantages of the invention are described below with reference to the appended drawings. The drawings show:

[0041] FIG. 1: a flow chart showing features of preferred embodiments of the method according to the invention for producing carrier particles;

[0042] FIG. 2: a flow chart showing features of preferred embodiments of the use of carrier particles produced according to the invention;

[0043] FIG. 3: photographs of rehydrated carrier particles compared to conventional carrier particles; and

[0044] FIG. 4: a schematic depiction of the method for an exemplary application of carrier particles produced according to the invention.

[0045] The invention is described below with exemplary reference to alginate-based cell carriers (carrier particles). Alginate beads, which have been loaded according to the invention with the lyoprotectant substance and subjected to freeze-drying, can for example be produced from commercially available alginate, which typically has a low viscosity due to relatively short chain lengths of the polymer macromolecules. Alternatively, use may be made of alginate having a higher viscosity, due to longer molecule chains, than the commercially available alginate. The selection of a specific alginate to be used is preferably made on the basis of the desired elasticity of the cell carrier during the cell culturing. The invention is not restricted to the use of alginate, but rather can also accordingly be carried out with other hydrogels, for example collagen, gellan or pectin. Embodiments of the invention are described below in particular with reference to examples for loading alginate with lyoprotectant substances, modifying alginate beads, dried alginate particles and/or rehydrated alginate beads, and the protocols for freeze-drying. Details of the use of the rehydrated alginate beads in the culturing of biological cells can be carried out as is known per se from conventional cell culturing.

[0046] FIG. 1 schematically shows the main steps of the production, according to the invention, of dried alginate particles. In step P1, alginate beads are produced in an aqueous suspension in a container. The alginate beads are produced for example by generating Na-alginate droplets using a nozzle and crosslinking the alginate droplets in an aqueous suspension liquid with an ionic precipitant, for example as described in [1]. A BaCl.sub.2 solution, for example, is used as precipitant. The size and size distribution of the alginate beads can be set by the dimension of the nozzle and the operating parameters of the nozzle. The crosslinked alginate beads form dimensionally stable alginate beads which are suspended in the suspension liquid.

[0047] The alginate beads are preferably functionalized after the precipitation by coating with tyramine and/or a protein, for example Matrigel. The coating takes place by sedimentation from the suspension liquid, or direct covalent coupling. The Matrigel coating affords advantages for the adhesion of cells in a subsequent cell culturing.

[0048] A lyoprotectant substance is already added to the suspension liquid while the alginate droplets are being dropped into the suspension liquid, or alternatively after the crosslinking and formation of the alginate beads. In the inventor’s tests, trehalose (100 mg/ml), poloxamer (trade name Pluronic F 68, 1 mg/ml), and/or sucrose (100 mg/ml) in an aqueous NaCl solution (0.9%), for example, were used as lyoprotectant substance. The suspended alginate beads are loaded with the lyoprotectant substance for example by storing the alginate beads in an aqueous solution containing the lyoprotectant substance, e.g. for at least one day.

[0049] Optionally, in step P2, the alginate beads loaded with the lyoprotectant substance are removed from the suspension. For example, the suspension liquid is decanted, leaving the alginate beads surrounded by residual liquid in the container. Alternatively, a screen is used for the removal.

[0050] If step P2 is not provided, in step P3 the freeze-drying of the alginate beads takes place immediately after the alginate beads are loaded with the lyoprotectant substance. The following phases are provided for this purpose.

[0051] Firstly, the alginate beads are cooled to an end temperature of e.g. -45° C. in a freezing phase over 150 min (approx. 0.4° C./min). During the freezing phase, a linear time-temperature function is for example applied.

[0052] Subsequently, a stabilization phase follows, in which the alginate beads are stored at the end temperature for a stabilization interval duration of 120 min. The stabilization phase has the advantage that the sample is completely frozen through with the frozen alginate beads before the drying phases are carried out.

[0053] A subsequent first drying phase has the function of a drying up phase, in which the suspension liquid is removed by means of sublimation. The first drying phase is performed for example in a lyophilizer with a cooling unit and a condenser. During the first drying phase, the end temperature, for example -45° C., is maintained, while the pressure is lowered following a linear time-pressure function over a duration of 10 min from atmospheric pressure down to a first negative pressure of 50 .Math.bar. Subsequently, in the first drying phase, the frozen sample is held at the end temperature and the first negative pressure for a stabilization duration of for example 80 hours.

[0054] In a subsequent second drying phase, a second negative pressure, which is lower than the first negative pressure and is for example 100 .Math.bar, is applied to the dried alginate particles. The lowering to the second negative pressure takes place with a linear time-pressure function over a duration of for example 300 min. During the second drying phase, the temperature of the dried alginate particles is equal to the end temperature or an increased temperature, for example room temperature (20° C.). After lowering to the second negative pressure, the dried sample is kept at the second negative pressure during the second drying phase for a stabilization duration of for example 20 hours.

[0055] Subsequently, a ventilation phase is provided, in which the dried alginate particles are transferred to a normal pressure according to a linear time-pressure function having a pressure increase interval of at least 1 min. The ventilation phase can be provided using air or an inert gas. Applying a relatively long pressure increase interval advantageously prevents damage to the dried alginate particles. If ventilating with air, the storage vessel is closed beforehand in the chamber, whereas when using an inert gas, the closure takes place after the ventilation.

[0056] Dry nitrogen or argon is preferably used as inert gas. The dried alginate particles are particularly preferably stored in the inert gas or in a vacuum. Practical tests gave a storability, e.g. at 4° C., of the dried alginate particles produced according to the invention without any loss of functionality for several months.

[0057] The method for freeze-drying P3 described by way of example can be modified, in terms of the temperatures and pressures set and the form of the time-pressure and time-temperature functions, on the basis of the specific conditions of application. For example, preparatory tests can be used to determine what time and pressure parameters deliver optimal drying results for a specific hydrogel, in particular alginate, sample.

[0058] As a result of the freeze-drying P3, the dried alginate particles are present as finished cell carriers. Because of the addition, according to the invention, of the lyoprotectant substance, the dried alginate particles have a spherical shape (see for example the photograph in FIG. 4), which advantageously also remains after rehydration. The dried particles can be joined together as a cake or form a pourable bulk material composed of individual particles. The formation of the pourable bulk material is promoted if a cohesion-reducing substance, for example polyethylene glycol, is added to the suspension of the alginate beads in addition to the lyoprotectant substance. Use is for example made of polyethylene glycol having the trade name PEG 600 at a concentration of 50 mg/ml in order to minimize adhesion of the dried alginate particles to one another.

[0059] Depending on the concentration of the lyoprotectant substance used, said substance may form residues on the surface of the dried alginate particles following the freeze-drying P3. Accordingly, as shown in FIG. 1, an optional step P4 may be provided for removing the lyoprotectant substance, in particular from the surfaces of the dried alginate particles, for example by screening or grinding.

[0060] An application of the dried alginate particles as cell carriers in the culturing of biological cells is shown schematically in FIG. 2. Firstly, in step K1, an aqueous culture medium is prepared in a bioreactor. The composition of the culture medium is selected on the basis of the specific application of the cell culture in a manner known per se. The alginate particles produced according to the invention are added to the culture medium. The dried alginate particles are metered in for example by means of weighing. Tests with dried alginate particles produced according to the invention showed that particles without polyethylene glycol loading initially lay on the surface of the culture medium and only sank into the culture medium after centrifugation, whereas with particles modified with polyethylene glycol, no floating on the culture medium was observed.

[0061] In the aqueous culture medium, in step K2, the rehydration of the alginate particles takes place. The dried alginate particles are converted into alginate beads by absorbing water from the culture medium, as shown for example in FIG. 3. Subsequently, in step K3, the cell culturing, cell incubation and/or cell storage of biological cells takes place in the culture medium with the alginate beads.

[0062] FIG. 3 shows light-microscopic images of alginate beads which, according to preferred variants of the invention, have been loaded with trehalose (A) or sucrose (B), freeze-dried and rehydrated, in comparison with alginate beads which have been freeze-dried and rehydrated without a lyoprotectant substance (C), and freshly produced alginate beads (D) in suspension (diameter approx. 400 .Math.m). The rehydrated particles (A, B) produced according to the invention advantageously clearly exhibit the same spherical shape as the suspended, untreated alginate beads (D). On the contrary, the untreated freeze-dried and rehydrated particles (C) (e.g. according to [1]) have a bumpy and irregular surface unlike the untreated alginate beads.

[0063] A further application of the dried alginate particles as cell carriers in the cell expansion of hiPS cells (human induced pluripotent stem cells) is schematically shown in FIG. 4. For a first expansion phase, dried alginate particles 1 are added to a first culture vessel 2, e.g. a suspension bioreactor having a volume of a few milliliters (e.g. 10 ml) to a few liters (e.g. 3 I). Alginate beads 3 (shown by way of example) are formed by rehydrating from the alginate particles 1. In addition, hiPSC cells are added to the culture vessel 2 from a vessel 4. The cell culturing then takes place under specified culture conditions, with the cells initially attaching and adhering to the microcarriers and then multiplying by a factor (e.g. sevenfold) over several days (e.g. 7 days). In a final step of the process, the alginate beads with adhered cells 5 are obtained; i.e., after they are obtained (e.g. by enzymatic treatment), the consumed microcarriers 6 and the multiplied hiPS cells 7 are present. These can subsequently be stored in a cell bank 10, further investigated and/or processed 9 (e.g. differentiation into cardiomyocytes) and/or transferred for direct use 8 (e.g. bioprinting).

[0064] The features of the invention disclosed in the above description and in the drawings and the claims can be significant, both individually and in combination or sub-combination, for carrying out the invention in the various configurations thereof.