MICROCARRIERS FOR CELL CULTURE

Abstract

A cell culture microcarrier bead is proposed. The microcarrier bead comprises a bead body having its surface flecked with plasmonic nanoparticles. In a second aspect, the invention relates to a cell culture reactor, containing a cell culture medium and the proposed microcarrier beads. A third aspect of the invention concerns a method for observing living cells on such microcarrier beads. Yet a further aspect of the invention relates to a method for packing nanoparticles on a carrier body.

Claims

1. A cell culture microcarrier bead, comprising a bead body having a surface, the surface flecked with plasmonic nanoparticles, the bead body having a diameter in the range from 50 μm to 1 mm. the bead body comprising a swollen or unswollen hydrogel.

2. The microcarrier bead as claimed in 1, wherein the plasmonic nanoparticles comprise plasmonic metal nanoparticles.

3. The microcarrier bead as claimed in claim 1, wherein the plasmonic metal nanoparticles comprise gold nanoparticles.

4. The microcarrier bead as claimed in claim 1, wherein the plasmonic nanoparticles are stabilized with a capping agent, e.g. citrate.

5. The microcarrier bead as claimed in claim 1, wherein the plasmonic nanoparticles are spherical.

6. The microcarrier bead as claimed in claim 1, wherein the plasmonic nanoparticles have a diameter in the range from 2 to 200 nm.

7. The microcarrier bead as claimed in claim 1, wherein the ratio of the diameter of the bead body to the diameter of the plasmonic nanoparticles lies in the range from 250 to 25000.

8. A cell culture reactor, containing a cell culture medium and microcarrier beads as claimed in claim 1.

9. A method for observing living cells on a microcarrier bead, the microcarrier bead comprising a bead body having a surface, the surface being flecked with plasmonic nanoparticles, the bead body having a diameter in the range from 50 μm to 1 mm. the bead body comprising a swollen or unswollen hydrogel, the method comprising: illuminating the microcarrier bead with probe light so as to excite localized surface plasmons in the plasmonic nanoparticles, and detecting light emitted from the plasmonic nanoparticles in response to the illumination.

10. The method for observing living cells as claimed in claim 9, wherein the detection is based on one or more of the following techniques: localized surface plasmon resonance (LSPR), Surface-enhanced Raman spectroscopy (SERS), fluorescence and second-harmonic generation.

11. A method for packing nanoparticles on a carrier body, comprising: attaching nanoparticles on a surface of said carrier body in a swollen state, reducing the volume of the carrier body and thereby causing the nanoparticles to move closer together.

12. The method as claimed in claim 11, wherein the nanoparticles are plasmonic nanoparticles, e.g. plasmonic metal nanoparticles.

13. The method as claimed in claim 11, wherein the carrier body comprises a microcarrier bead for cell culture.

14. The method as claimed in claim 11, wherein the carrier body comprises a hydrogel and wherein said volume reduction is effected by at least partial dehydration of the hydrogel.

15. A method for culturing living cells on a microcarrier bead, the microcarrier bead comprising a bead body having a surface, the surface being flecked with plasmonic nanoparticles, the bead body having a diameter in the range from 50 μm to 1 mm. the bead body comprising a swollen or unswollen hydrogel, the method comprising stimulating a release of cells from the microcarrier bead by exciting localized surface plasmon resonance in said plasmonic nanoparticles.

16. The method as claimed in claim 15, wherein said release of cells is stimulated by plasmonic heating of said nanoparticles.

17. The microcarrier bead as claimed in 1, wherein the plasmonic nanoparticles comprise plasmonic metal nanoparticles and wherein the plasmonic metal nanoparticles comprise gold nanoparticles.

18. The microcarrier bead as claimed in claim 17, wherein the plasmonic nanoparticles are stabilized with a capping agent comprising citrate.

19. The microcarrier bead as claimed in claim 18, wherein the plasmonic nanoparticles are spherical.

20. The microcarrier bead as claimed in claim 19, wherein the plasmonic nanoparticles have a diameter in the range from 2 to 200 nm and wherein the ratio of the diameter of the bead body to the diameter of the plasmonic nanoparticles lies in the range from 250 to 25000.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings illustrate several aspects of the present invention and, together with the detailed description, serve to explain the principles thereof. In the drawings:

[0027] FIG. 1: is a schematic of a cell culture microcarrier having its surface coated with plasmonic nanoparticles;

[0028] FIG. 2: is an illustration of a process for attaching plasmonic nanoparticles on a microcarrier for cell culture.

[0029] FIG. 3: is chart of the size distributions of plasmonic nanoparticles (left-hand side) and microcarriers (right-hand side) used in examples of implementation of the invention;

[0030] FIG. 4: shows SEM images of hydrogel microcarriers covered with different concentrations of gold nanoparticles and then dried;

[0031] FIG. 5: shows SEM images of microtomed slices of AuNP-coated microcarriers, focused on the interface between fused microcarriers, with magnification increasing from (i) to (iv);

[0032] FIG. 6: shows optical microscopy images of naked MCs in dry state (a), of naked MCs in water (c), of AuNP-coated MCs in water, and the corresponding observed size distributions (d);

[0033] FIG. 7: shows a photography of suspensions of Au nanoparticle coated microcarriers with increasing coverage (a)), a graph of the corresponding absorbance spectra (b)) and a graph of the peak intensities and positions (c));

[0034] FIG. 8: is a chart of the evolution of glucose and lactate concentrations in cell cultures on naked microcarriers and on plasmonic-nanoparticle-coated microcarriers.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0035] FIG. 1 schematically illustrates a cell culture microcarrier bead 10. The microcarrier bead 10 comprises a bead body 12 (e.g. a conventional, commercially available MC) having its surface flecked with plasmonic nanoparticles 14. Also illustrated are cells 16 that use the microcarrier bead 10 as a growth surface.

[0036] Regarding the integration of sensing functions, such as, e.g., cell health monitoring and/or biomarker reporting, into microcarrier beads, it is a key requirement that the modification of MCs does not have an uncontrolled adverse impact on cell adhesion and proliferation and that the sensing function(s) are compatible with the environment, in which the MCs are to be used. Therefore, when microcarrier beads with attached plasmonic nanoparticles are to be used, comparative tests should be carried out in order to assess whether (and, if necessary, to what extent) the presence of the plasmonic nanoparticles changes the behaviour of the cultured cells.

[0037] In the examples discussed below, it is shown that the integration of high-density assemblies of gold nanoparticle on the surface of dextran-based MCs (commercially available Cytodex-1™ microcarriers based on a cross-linked dextran matrix that is substituted with positively charged N,N-diethylaminoethyl, DEAE, groups) retain the cell-expansion and cell-migration capabilities on par with those of the uncoated (unmodified) MCs. It is further demonstrated that the gold-nanoparticle-flecked MCs are suitable for plasmonic sensing. The results show that gold nanoparticles are an excellent choice to integrate onto microcarriers, given the biocompatibility of gold particles, ease of sensing using their unique optical, spectroscopic properties, while maintaining stability in complex environment.

[0038] As illustrated in FIG. 2, the attachment of gold nanoparticles 114 on MCs 112 may be effected by electrostatic self-assembly, taking advantage of the presence of positive charges within the polymer matrix of the MCs. FIG. 2 more specifically illustrates electrostatic self-assembly of citrate-stabilized gold nanoparticles 114 (see enhanced detail) on a positively charged hydrogel MC 112. Electrostatic self-assembly processes are known as such but, to the best knowledge of the inventors, have not been previously tried on MCs. Irreversible monolayer attachment of charged colloids to oppositely charged surfaces may be considered as a random-sequential adsorption (RSA) process that results in a theoretical maximum surface coverage of 54.7% (jamming limit), although the practically attained surface coverages are typically significantly lower.

[0039] MCs flecked with plasmonic nanoparticles are useful, e.g., for sensing based on surface-enhanced Raman spectroscopy (SERS), taking advantage of the enhanced electromagnetic fields arising in close proximity of nanostructured surface. The magnitude of the enhancement depends on the geometric attributes of the features and their assemblies, and is non-linear with decreasing separation between neighboring nanoparticles. High electromagnetic fields at junctions between nanoparticles are termed “gap hot-spots”. The likelihood of occurrence of such hot spots increases with nanoparticle density. It may thus be particularly advantageous to assemble the plasmonic nanoparticle on hydrogel-based microcarriers in conditions (e.g., temperature, pH, etc.) wherein the latter are heavily swollen. By making the MCs shrink (by changing the environmental conditions), the mean distance between the nanoparticles attached to the surface is reduced, which favours the occurrence of the above-mentioned hot spots.

Examples

[0040] DEAE-dextran MCs (Cytodex-1™, purchased from Sigma-Aldrich) were functionalized with citrate-stabilized gold nanoparticles by electrostatic interaction, as represented in FIG. 2. MilliQ water (18.2 MΩcm, Millipore) was used for the preparation of the solutions and for all rinses. Citrate-stabilized gold nanoparticles (AuNPs) were prepared according to the Turkevich method, by reduction of chloroauric acid (HAuCl.sub.4, Sigma-Aldrich) with sodium citrate (Na.sub.3C.sub.6H.sub.5O.sub.7, Sigma-Aldrich). The obtained AuNPs were substantially spherical with an average size of 12 nm.

[0041] The prepared colloidal suspension was stored at 4° C. and taken out from the refrigerator 12 hours before being used. Eppendorf Safe-Lock Tubes 2 ml, Conical Bottom Centrifuge Tubes 50 ml, Silicon Wafers (Siegert Wafer) and Glass Wafers (Menzel™ Microscope Coverslips) were used as containers and supports for characterization, respectively.

[0042] The size of the AuNPs was measured by Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). FIB-SEM images of the AuNPs were obtained using Helios NanoLab™ 650 microscope operating at an accelerating voltage of 20 kV and a distance of 4 mm. The secondary electron detector was used. The AuNPs were adsorbed on positively charged silicon surface functionalized with 3-aminopropyl trimethoxy silane, then the size of the AuNPs was measured.

[0043] The size of the MCs was measured by optical microscopy. Images of the MCs were obtained using Olympus Microscope BX51. Two objectives (Olympus; ×10, ×50) were used to focus. Images were acquired using OLYMPUS Stream Image Analysis Software.

[0044] FIG. 3 shows the size distributions of the AuNPs (left) measured by FIB-SEM and of the AuNP-flecked MCs (right) in water.

[0045] Dynamic Light Scattering (DLS) was used to determine hydrodynamic size and zeta potential of the gold nanoparticles. The machine used was the MALVERN ZETASIZER NANO ZS. Disposable Folded Capillary Zeta Cell (MALVERN) was used for the measurement of both parameters. The pH of the AuNP solution was equal to 5.8. The sample was prepared as follows: 100% and 33% diluted solution of gold nanoparticles were injected inside two different disposable cells (for comparison). The zeta potential of the gold nanoparticles was determined by DLS to be equal to −34±10 mV at pH 6. The zeta potential is negative due to the capping agent (citrate) which is negatively charged at that pH value. The microcarriers being too large, they could not be characterized by DLS. However, it is known that DEAE-dextran is positively charged at almost all pH due to its DEAE-groups. Consequently, the gold nanoparticles were expected to cover the surface of the microcarriers. Cytodex-1 is a cross-linked hydrogel having positive groups throughout the entire matrix. Thus, the question arises whether gold nanoparticles penetrate inside the microcarriers and functionalize the internal surfaces (pores). However, as detailed below, the characterization of external and internal structure reveals the distribution of gold nanoparticles to the outer surface only. DEAE-dextran microcarriers swell when added into any medium. Consequently, the surface area available for interaction with gold nanoparticles changes according to the medium used.

[0046] The distribution and number of gold nanoparticles per microcarrier was investigated by SEM. FIG. 4 shows SEM images of MCs covered with different concentrations of gold nanoparticles and then dried. The shrinking of the MCs during the drying process results in a wrinkled surface and a denser packing of the AuNPs. The AuNP coverage increases from left to right (shown at two magnifications). The transition from low to high concentration is easily observable. At saturated coverage, the microcarriers were found to be fully covered with nanoparticles, while at lower particle coverages, the nanoparticles show homogeneous, yet patchy coverage.

[0047] To investigate the questions whether gold nanoparticles penetrate into the pores of the MCs, the AuNP-flecked MCs were pelleted from suspension, then dried in an oven at 80° C., sliced by ultra-microtomy, and characterized by SEM and Nano-SIMS (nanoscale secondary ion mass spectrometry). The process of oven drying was found to fuse the microcarrier interfaces, as can be observed in FIG. 4. Complete separation of microcarriers could not be achieved, even though better results were obtained than when drying in the oven. The nanoparticles could be seen in SEM to be restricted to the interface between microcarriers, with no evidence of an internal distribution within the MC (see FIG. 5). Nano-SIMS measurements of the elemental distribution of .sup.197Au on microtome slice confirm this finding. Therefore, the NP-covered MC can be represented by a simple geometric model, with AuNPs evenly distributed on the exterior of the swollen MCs.

[0048] FIG. 6 shows the size distributions (d) of naked MCs in dry state (a), of naked MCs in water (c) and of AuNP-coated MCs. The swelling from dry to fully hydrated state (+260% in terms of diameter) in water is higher than the swelling that is achieved when putting dry MCs into a AuNP suspension (+115% in terms of diameter). The lower swelling in AuNP suspension can be the result of screening of electrostatic repulsion resulting from high ionic strength.

[0049] Taking RSA as a model for the electrostatic assembly process of the AuNPs on the MC surface, the concentration of NPs (in NPs per MC) necessary to reach the jamming limit can be calculated as:

[00001]$\begin{array}{cc}{\left[NP\right]}_{\mathrm{ideal}}=\frac{54.7\%.Math.S_{\mathrm{MC}}}{S_{\mathrm{NP}}}=2.2{\left(\frac{R}{r}\right)}^2,& \left(\mathrm{Eqn}.1\right)\end{array}$

where S.sub.MC=4πR.sup.2 is the surface area of the MCs with R being the MC radius and S.sub.NP=πr.sup.2 is the footprint area of the gold nanoparticles with r being the NP radius. With r=6 nm and R=86 μm, [NP].sub.ideal=4.5.Math.10.sup.8.

[0050] To coat the MCs with gold nanoparticles, the nanoparticle concentrations in the suspension ([NP].sub.ution) was increased systematically in relation to the nanoparticle concentrations corresponding to a monolayer coverage ([NP].sub.ideal, Eqn 1). The concentration of nanoparticles in solution ([NP].sub.ution) is defined as the number of nanoparticles in solution available per microcarrier and is calculated by dividing the total number of nanoparticles by the total number of microcarriers present in the same volume. The total number of nanoparticles in the suspension can be estimated from the ratio of the mass of gold in solution to the mass of one gold nanoparticle. The mass of gold in solution is known from the molar concentrations of gold salt used during synthesis, all of which is assumed to be reduced to gold. The mass per gold particle is obtained by approximating the NPs as spheres, with the diameter corresponding to the peak of the size distribution (cf. FIG. 3), and assuming the density of gold NPs to be the same as bulk gold. The number of MCs per unit volume was derived from the manufacturer's specification of the number of MCs per gram of dry weight, which was 6.8.Math.10.sup.6 g.sup.−1 for Cytodex™-1, and the mass of dry powder employed.

[0051] Cytodex™-1 MCs coated with AuNPs are easily distinguished from Cytodex-1 without any coating, since the naked MCs are transparent in water, the coated MCs appear purple with intensity depending on nanoparticle coverage (FIG. 7 a)). The absorbance spectra of the microcarriers coated with Au nanoparticles a systematic increase in the peak intensity of the localized surface plasmon resonance band of gold particles, as a function of nanoparticle coverage (FIG. 7 b) and c)). The increase in peak intensity is also associated with a red shift of the peak wavelength (FIG. 7 b) and c)). Both changes plateau at the saturated coverage of gold particles (FIG. 7 c)). The red shift in peak position is in line with expectations for plasmonic coupling between adjacent particles, the opportunity for which increases with particle coverage. Saturated coverage was achieved when the nanoparticle concentration amounted to three or more times [NP].sub.ideal. UV-visible spectra of the microcarriers were obtained using a TECAN Infinite M1000 PRO plate reader and were recorded in the 250 to 1000 nm range. Greiner CELLSTAR™ 96-well plate was used as plate reader. The sample was prepared as follows: 200 μl of each MCs suspension (C0-C9) were added into the microplate wells. The table below indicates the reagents used for functionalization of microcarriers with AuNPs, namely the mass of Cytodex-1, the volume of AuNPs suspension (produced as indicated above) and the volume of water added to reach a total volume of 2 ml each time.

TABLE-US-00001 Sample Mass of Volume of AuNP Volume of name MCs [mg] solution [ml] water [ml] C.sub.0 10 0.000 2.000 C.sub.1 10 0.060 1.940 C.sub.2 10 0.120 1.880 C.sub.3 10 0.240 1.760 C.sub.4 10 0.480 1.520 C.sub.5 10 0.960 1.040 C.sub.6 10 1.920 0.080 C.sub.7 5 1.920 0.080 C.sub.8 2.5 1.920 80 C.sub.9 1 1.536 464

[0052] The different masses of commercial MC powder were weighted directly in nine different Eppendorf tubes. The respective volumes of AuNP suspension and water were then added. The MCs were incubated for 3 hours. After incubation, the MCs had sedimented and the supernatant was removed. The MCs were then washed three times by: [0053] adding water in the Eppendorf tube and pipetting up and down to wash all the MCs in the solution; [0054] centrifuging at 1300 g during 2 minutes; and [0055] removing the supernatant.

[0056] The AuNP-coated MCs were then characterized as to their spectra.

[0057] Inelastic scattering properties were evaluated by Raman spectroscopy. Raman Scattering spectra were recorded in the 100 to 4000 cm.sup.−1 extended range using Renishaw inVia™ Raman microscope equipped with a high-power near-IR laser diode working at 785 nm and a visible laser diode working at 633 nm. Before acquisition of the spectra, an optical microscope (Olympus; objective, ×50 L) was used to focus the laser beam. The laser output power was 0.9 mW (10%). For each spectrum, 6 accumulations of 5 seconds were recorded. To ensure a representative characterization of surfaces, multiple measurements were taken on both different parts of the same microcarrier and different microcarriers. Results were analyzed using WiRE™ and OriginLab™ software. Spectra acquired were smoothed and normalized. The sample was prepared as follows: some MCs were placed on a carbon tab.

[0058] Cell culture tests were carried out with AuNP-coated MCs (Cytodex™-1) and the uncoated version thereof. As shown in FIG. 8, cell kinetic growth was monitored by measuring the time evolution of the concentration in glucose and lactate on a daily basis. Glucose is the main source of energy for the cell and lactate is one of the metabolite by-products of the glucose metabolism. Glucose and lactate concentrations are thus important indicators of cell growth and metabolic activity. About 50-100% of glucose is usually converted to lactate in cell culture fermentation. Cells on AuNP-coated Cytodex™-1 MCs consumed glucose in the same way as those on the uncoated Cytodex™-1 MCs. The consumption rate of glucose slowed down when glucose became limited and the production rate of lactate decreased consequently. No significant difference between commercial Cytodex™-1 and AuNPs-coated Cytodex™-1 MCs was noted.

[0059] Before cell culture, Cytodex™-1 MCs should be swelled in PBS (phosphate-buffered saline) and autoclaved at 120° C. to sterilize them. However, the Cytodex-1 MCs were not autoclaved before attachment of gold nanoparticles. Since after AuNP attachment potentially impacts the interaction of gold nanoparticles with microcarriers, cell culture was performed on both autoclaved and non-autoclaved AuNP-coated Cytodex™-1.

[0060] Quantification of cell viability was effected by fluorescent microscopy. The cells (human mesenchymal stem cells) were mixed with Calcein AM and Ethidium Homodimer-1 (EthD-1) molecules to stain live (green) and dead (red) cells, respectively. It was found that cells colonized AuNP-coated Cytodex™-1 in a larger extent when they were not autoclaved. Hence, autoclaving has an impact on the microcarriers that inhibits adhesion of cells. However, the interaction between AuNPs and Cytodex™-1 MCs did not seem to be affected: the microcarriers were still opaque after autoclaving, confirming the presence of AuNPs. Without wanting to be bound by theory, the inventors indicated that one reason for the lower cell adhesion might be the break of the electrostatic interaction between the gold nanoparticles and the citrate (stabilizer of the gold nanoparticles) due to high temperature. The removal of the negatively charged citrate molecules would change the overall charge on the surface of the microcarrier and thus affect the interaction with the cell.

[0061] It was furthermore tested whether cells cultured on commercial microcarriers would migrate to plasmonic-nanoparticle-flecked microcarriers. The migration assay comprised adding (non-autoclaved) AuNP-coated Cytodex™-1 into a cell culture on naked but otherwise identical Cytodex™-1 microcarriers.

[0062] 2 ml of Cytodex™-1 suspension (20 g/l) and 25 ml of cell culture medium were added into a 125 ml Erlenmeyer flask. The MCs were kept in that container during 1 hour at 37° C. and 5% CO.sub.2. Cells were detached and harvested from their container flasks by trypsinization. They were added into the Erlenmeyer already containing the microcarriers (0.8.Math.10.sup.5 cells/ml). Cells and MCs were incubated in the cell culture medium to allow adhesion of the cells on the MCs. After 1 hour, cells anchored on microcarriers were put in agitation at 70 RPM (37° C.; 5% CO.sub.2). Samples of cell culture medium were withdrawn on a daily basis to measure glucose and glutamine consumption, as well as lactate, ammonium and lactate dehydrogenase production. Cell viability was assessed by fluorescence microscopy. To do this, cells were mixed with DAPI, Calcein AM and Ethidium Homodimer-1 (EthD-1) molecules to stain cells, live cells and dead cells, respectively.

[0063] On day 4, 50% of the cell culture medium was changed. Microcarriers coated with gold nanoparticles) were added to the Cytodex™-1 culture flask on day 4. After 1 hour of incubation, agitation was restarted (70 RPM). Quantification of cells was made on a daily basis with fluorescent microscopy. The cells were mixed with DAPI to stain cells. Cells were able to move towards the new carriers, confirming the capability of the modified microcarriers to support cell-expansion functions.

[0064] To summarize, the optical properties of the plasmonic-nanoparticle-coated microcarriers correlate with the gold nanoparticle coverage and indicate strong plasmonic coupling at saturated coverage. The plasmonic-NP-coated MCs open the possibility to detect smaller organic molecules using SERS or other plasmonic sensing techniques. Comparison of the plasmonic-NP-coated MCs with their un-coated counterparts showed similar cell growth kinetics, and migration of human mesenchymal stem cells, giving first evidence of the retention of cell-expansion functionality. The plasmonic microcarriers reported here, pave the way forward for plasmonic sensing of changes at the interface between microcarriers and biological media, and for monitoring changes to the biological cells at the different phases of cell-expansion, and to potentially enable plasmonic release of cells by plasmonic heating effects.

[0065] While specific embodiments have been described herein in detail, those skilled in the art will appreciate that various modifications and alternatives to those details could be developed in light of the overall teachings of the disclosure. Accordingly, the particular arrangements disclosed are meant to be illustrative only and not limiting as to the scope of the invention, which is to be given the full breadth of the appended claims and any and all equivalents thereof.