Microcarriers for cell culture

Abstract

Microcarrier beads for cell culture are proposed. The microcarrier beads consist of a bead body having a surface dotted 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 relates to a method for observing living cells on such microcarrier beads. Yet a further aspect of the invention relates to a method for loading nanoparticles onto a carrier body.

Description

[Technical field]

[0001] The present invention relates generally to microcarriers for cell culture, methods for making such microcarriers and uses thereof. [Background technology]

[0002] Microcarriers (MCs), measuring tens of microns in size, have been used as growth surfaces for 3D cell culture, allowing cell growth in a controlled environment within a bioreactor with the advantages of better nutrient availability, expanded surface area, and automated and high-throughput harvesting.

[0003] Although research in this area has focused on more novel MCs designed to provide chemical and mechanical stimuli that affect cell proliferation, maintain stem cell stemness, promote differentiation of stem cells into desired cell types, and allow easy release of cells, MCs made from organic and inorganic materials and exhibiting different surface chemistries are commercially available. Summary of the Invention [Problem to be solved by the invention]

[0004] Beyond the above goals, a further significant advancement for the use of MCs for cell proliferation would be the integration of sensing capabilities that could help monitor cell health and / or report biomarkers of interest during cell culture. [Means for solving the problem]

[0005] According to a first aspect of the present invention, a cell culture microcarrier bead is proposed, which is made of a bead body having a surface dotted with plasmonic nanoparticles (plasmonic nanoparticle-coated).

[0006] As used herein, the term "nanoparticle" refers to a particle having a surrounding interfacial layer and a characteristic size in the sub-micrometer range, e.g., a diameter in the range of 1-500 nm. Preferably, plasmonic metal nanoparticles have a diameter in the range of 2-200 nm. In this document, unless the context is inconsistent, the term "diameter" refers to the smallest distance that can be formed between two opposing parallel faces that are tangent to the convex hull of the object of interest. Diameter measurements can be made, for example, by scanning electron microscope (SEM), transmission electron microscope, or scanning probe microscope (SPM).

[0007] Preferably, the plasmonic nanoparticles comprise or consist of metal nanoparticles. Such plasmonic metal nanoparticles may comprise or consist of, for example, gold, silver or aluminum nanoparticles, although other plasmonic metals or alloys are not excluded. For example, the plasmonic nanoparticles may be functionalized so that they specifically bind to receptors on cells.

[0008] The beaded bodies may be made of a hydrogel matrix in a swollen (hydrated) or non-swollen (dehydrated) state. Such a hydrogel matrix may comprise or consist of, for example, polysaccharides (e.g. agarose, dextran, etc.), elastin-like polypeptides (ELPs), etc. The beaded bodies may consist of such a hydrogel matrix or, alternatively, have a (solid) core surrounded by a hydrogel matrix.

[0009] The bead body preferably carries positively charged groups distributed within its volume, in which case attachment of the plasmonic nanoparticles to the bead body can be effected by electrostatic assembly.

[0010] Preferably, the microcarrier beads carry plasmonic nanoparticles only on the surface of the bead body, i.e. not inside the bead body, or at least not deep inside the bead body, "deep" in this context means more than 0.3 x R from the surface, where R refers to the radius of the bead body.

[0011] The plasmonic nanoparticles can be stabilized with a capping agent, for example, citric acid.

[0012] Plasmonic nanoparticles may be of any shape, e.g. spheres, stars, rods, cubes, etc. In case of spherical nanoparticles, their diameter is preferably 100 nm or less. Nanoparticles of other shapes, e.g. nanostars, nanorods, etc., should be of larger size. In case the nanoparticles are nanorods, the cross-sectional diameter should preferably be in the range of 2-200 nm, while the length of the nanorods may be longer.

[0013] The body of the microcarrier beads preferably has a diameter in the range of 50 μm to 1 mm. The ratio of the diameter of the bead body to the diameter of the plasmonic metal nanoparticles is preferably in the range of 250 to 50000, more preferably in the range of 250 to 25000. In the case of bead bodies with a hydrogel matrix, the indicated diameter ranges may refer to the diameter in the dehydrated state, or in the hydrated state (e.g. for use in cell culture medium) or any intermediate state.

[0014] In a second aspect, the present invention relates to a cell culture reactor containing a cell culture medium and the microcarrier beads described herein.

[0015] A further aspect of the present invention relates to a method for observing (in vitro) living cells on microcarrier beads as described herein, said method comprising: illuminating the microcarrier beads with probe light to excite surface plasmons in the plasmonic metal nanoparticles; detecting light emitted from said plasmonic metal nanoparticles in response to illumination.

[0016] The detection is preferably based on one or more of the following techniques: (localized) surface plasmon resonance (SPR), surface enhanced Raman spectroscopy (SERS), fluorescence and second harmonic generation. SPR techniques measure changes in the absorbed wavelength or some other property of the detected light, which is indicative of local refractive index variations induced by cellular reactions, such as the interaction of a cell with another biological entity (e.g. an antigen), cell division, etc.

[0017] Yet a further aspect of the present invention relates to a method for loading nanoparticles onto a carrier body, which may be particularly useful in loading plasmonic nanoparticles onto microcarriers for cell culture, but may also be used for other purposes. - depositing nanoparticles onto a surface of the carrier body while the carrier body is in a swollen state; Reducing the volume of the carrier body, thereby bringing the nanoparticles closer together.

[0018] The attachment process itself may be effected using electrostatic attraction between the carrier body and the nanoparticles. It will be appreciated that such electrostatic assembly can be considered as a random-sequential adsorption (RSA) process with a theoretical maximum coverage. The coverage achieved in experiments is typically significantly lower than the theoretical maximum coverage, especially due to time limitations. It will therefore be appreciated that the method allows for an increase in the density of nanoparticles by attaching the nanoparticles when the carrier body is swollen and then shrinking the carrier body. A shorter average distance between nanoparticles in the shrinking state may be beneficial in practical applications, such as plasmonic sensing. Alternatively, the method can be used to shorten the attachment process for a given target density of nanoparticles.

[0019] As indicated above, the nanoparticles may be plasmonic nanoparticles, such as plasmonic metal nanoparticles.Furthermore, the carrier body may be made of microcarrier beads for cell culture.

[0020] Preferably, the carrier body comprises a hydrogel matrix and the volume reduction is brought about by (at least partial) dehydration of the hydrogel.

[0021] It will be appreciated that the presence of plasmonic nanoparticles on microcarrier beads can be used, for example, for cell recovery. For example, cell release from the microcarrier beads can be stimulated by exciting localized surface plasmon resonance in the plasmonic nanoparticles. Specifically, cell release can be stimulated by plasmonic heating of the nanoparticles.

[0022] The accompanying drawings illustrate several aspects of the invention and, together with the detailed description, serve to explain their principles. [Brief description of the drawings]

[0023] [Figure 1] FIG. 1 is a schematic diagram of a microcarrier for cell culture having a surface coated with plasmonic nanoparticles. [Diagram 2] FIG. 2 is a diagram illustrating a process for attaching plasmonic nanoparticles onto microcarriers for cell culture. [Diagram 3] FIG. 3 is a diagram of the size distribution of plasmonic nanoparticles (left) and microcarriers (right) used in a practical example of the present invention. [Figure 4] FIG. 4 shows SEM images of hydrogel microcarriers coated with various concentrations of gold nanoparticles and then dried. [Diagram 5]FIG. 5 shows SEM images of microtome sections of AuNP-coated microcarriers focused on the interface between fused microcarriers at increasing magnifications from (i) to (iv). [Figure 6] FIG. 6 shows optical microscopy images of bare MCs in the dry state (a), bare MCs in water (c), and AuNP-coated MCs in water (d) and the corresponding observed size distributions. [Figure 7] FIG. 7 shows photographs (a)) of suspensions of microcarriers coated with Au nanoparticles with increasing coverage, corresponding absorbance spectrum graphs (b)) and peak intensity and peak position graphs (c)). [Figure 8] FIG. 8 is a graph showing the evolution of glucose and lactate concentrations during cell culture on bare microcarriers and on microcarriers coated with plasmonic nanoparticles. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Figure 1 illustrates diagrammatically a microcarrier bead 10 for cell culture. The microcarrier bead 10 is made of a bead body 12 (e.g., conventional commercially available MC) having a surface dotted with plasmonic nanoparticles 14. Also illustrated is a cell 16 that uses the microcarrier bead 10 as a growth surface.

[0025] With regard to the integration of sensing functions, such as cell health monitoring and / or biomarker reporting, into microcarrier beads, key requirements are that the modification of the MC does not have uncontrollable detrimental effects on cell attachment and proliferation, and that the sensing function is compatible with the environment in which the MC will be used. Therefore, when using microcarrier beads with attached plasmonic nanoparticles, comparative studies should be performed to assess whether (and if so, to what extent) the presence of the plasmonic nanoparticles alters the behavior of cultured cells.

[0026] In the examples discussed below, it is shown that the integration of high-density assemblies of gold nanoparticles onto the surface of dextran-based MCs (commercially available Cytodex-1™ microcarriers, based on a cross-linked dextran matrix substituted with positively charged N,N-diethylaminoethyl (DEAE) groups) retains cell proliferation and migration capabilities comparable to those of uncoated (unmodified) MCs. It is further demonstrated that gold nanoparticle-studded MCs are suitable for plasmonic sensing. Given the biocompatibility, ease of sensing using the inherent optical and spectroscopic properties of gold particles, as well as maintaining stability in complex environments, the results indicate that gold nanoparticles are an excellent choice for integration onto microcarriers.

[0027] As illustrated in Fig. 2, the attachment of gold nanoparticles 114 onto MC 112 can be brought about by electrostatic self-assembly, taking advantage of the presence of positive charges within the polymer matrix of MC. Fig. 2 more specifically illustrates the electrostatic self-assembly of citrate-stabilized gold nanoparticles 114 (see enlarged detail) onto a positively charged hydrogel MC 112. The electrostatic self-assembly process is known per se, but to the inventors' knowledge, has not been attempted before for MC. The irreversible monolayer attachment of charged colloids to an oppositely charged surface can be thought of as a random sequential adsorption (RSA) process, which results in a theoretical maximum surface coverage of 54.7% (jamming limit), although the surface coverage achieved in practice is typically significantly lower.

[0028] Plasmonic nanoparticle-doped MCs are useful, for example, for sensing based on surface-enhanced Raman spectroscopy (SERS), which takes advantage of the enhanced electromagnetic fields that arise in close proximity to the nanostructured surface. The magnitude of the enhancement depends on the geometrical attributes of the features and their assembly, and becomes nonlinear as the separation between adjacent nanoparticles decreases. High electromagnetic fields at the junctions between nanoparticles are referred to as "gap hot-spots". The likelihood of the appearance of such hot-spots increases with nanoparticle density. It may therefore be particularly advantageous to assemble plasmonic nanoparticles on hydrogel-based microcarriers under conditions (e.g., temperature, pH, etc.) that result in a large swelling of the hydrogel-based microcarriers. Shrinking the MC (by changing the environmental conditions) reduces the average distance between the nanoparticles attached to the surface, facilitating the appearance of the above-mentioned hot-spots. EXAMPLES

[0029] DEAE-dextran MC (Cytodex-1™, purchased from Sigma-Aldrich) was functionalized with citrate-stabilized gold nanoparticles by electrostatic interactions, as shown in Figure 2. Milli-Q water (18.2 MΩcm, Millipore) was used for solution preparation and all rinsing. Citrate-stabilized gold nanoparticles (AuNPs) were prepared by the Turkevich method via reduction of chloroauric acid (HAuCl4, Sigma-Aldrich) with sodium citrate (Na3C6H5O7, Sigma-Aldrich). The resulting AuNPs were essentially spherical with an average size of 12 nm.

[0030] Prepared colloidal suspensions were stored at 4° C. and removed from the refrigerator 12 h before use. 2 mL Eppendorf safe-lock tubes, 50 mL conical-bottom centrifuge tubes, silicon wafers (Siegert Wafer) and glass wafers (Menzel™, microscope cover slips) were used as containers and supports, respectively, for characterization.

[0031] 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 a Helios NanoLab™ 650 microscope operating at an accelerating voltage of 20 kV and a distance of 4 mm. A secondary electron detector was used. The AuNPs were adsorbed onto a positively charged silicon surface functionalized with 3-aminopropyltrimethoxysilane, and then the size of the AuNPs was measured.

[0032] The size of MCs was measured by optical microscopy. Images of MCs were obtained using an Olympus microscope BX51. Two objective lenses (Olympus; x10, x50) were used for focusing. Images were acquired using OLYMPUS Stream image analysis software.

[0033] FIG. 3 shows the size distribution of AuNPs (left) and AuNP-doped MC (right) in water measured by FIB-SEM.

[0034] Dynamic Light Scattering (DLS) was used to determine the hydrodynamic size and zeta potential of the gold nanoparticles. The instrument used was a MALVERN ZETASIZER NANO ZS. A disposable Folded Capillary Zeta Cell (MALVERN) was used for the measurement of both parameters. The pH of the AuNP solution corresponded to 5.8. Samples were prepared as follows: 100% and 33% diluted solutions of gold nanoparticles were injected inside two different disposable cells (for comparison). The zeta potential of the gold nanoparticles was determined by DLS to correspond to -34 ± 10 mV at pH 6. The zeta potential is negative due to the capping agent (citric acid), which is negatively charged at that pH value. The microcarriers were too large to characterize them by DLS. However, DEAE-dextran is known to be positively charged at almost all pH due to its DEAE groups. As a result, it was expected that the gold nanoparticles would cover the surface of the microcarriers. Cytodex-1 is a cross-linked hydrogel with cationic groups throughout the matrix. Thus, the question arises as to whether gold nanoparticles penetrate inside the microcarriers and functionalize the internal surface (pores). However, as detailed below, characterization of the external and internal structure only reveals the distribution of gold nanoparticles relative to the outer surface. DEAE-dextran microcarriers swell when added to any medium. As a result, the surface area available for interaction with gold nanoparticles varies depending on the medium used.

[0035] The distribution and number of gold nanoparticles per microcarrier was investigated by SEM. Figure 4 shows SEM images of MCs coated with various concentrations of gold nanoparticles and then dried. The shrinkage of the MC during the drying process leads to a wrinkled surface and dense packing of AuNPs. The coverage of AuNPs increases from left to right (shown at two magnifications). The transition from low to high concentration is easily observed. At saturation coverage, the microcarriers were found to be completely covered with nanoparticles, whereas at lower particle coverage, the nanoparticles show a uniform but sparse coverage.

[0036] To investigate the question of whether gold nanoparticles penetrate into the pores of the MC, AuNP-studded MC were pelleted from the suspension, then dried in an oven at 80 °C, sectioned in an ultramicrotome, and characterized by SEM and Nano-SIMS (nanoscale secondary ion mass spectrometry). As can be observed in Figure 4, the process of drying in the oven was found to fuse the contact surfaces of the microcarriers. Even though better results were obtained than when drying in the oven, complete separation of the microcarriers could not be achieved. In the SEM, the nanoparticles could be seen restricted to the contact surfaces between the microcarriers, with no evidence of internal distribution within the MC (see Figure 5). On the microtome sections, 197 Nano-SIMS measurements of the elemental distribution of Au support this result. Thus, the NP-covered MC can be represented by a simple geometrical model with AuNPs uniformly distributed on the outer surface of the swollen MC.

[0037] Figure 6 shows the size distribution of bare MC in dry state (a), bare MC in water (c) and MC coated with AuNP (d). The swelling from dry to fully hydrated state in water (+260% in diameter) is greater than the swelling achieved when dry MC is placed in AuNP suspension (+115% in diameter). The reduced swelling in AuNP suspension may be a result of the shielding of electrostatic repulsion due to high ionic strength.

[0038] Taking RSA as a model for the electrostatic assembly process of AuNPs on the MC surface, the concentration of NPs (in NPs per MC) required to reach the packing limit is

[0039]

number

[0040] where S MC =4πR 2 (R is the radius of the MC) is the surface area of ​​the MC, and S NP=πr 2 (r is the radius of the NP) is the footprint area of ​​the gold nanoparticle. For r = 6 nm and R = 86 μm, [NP] ideal =4.5×10 8 It is.

[0041] To coat the MCs with gold nanoparticles, the nanoparticle concentration in the suspension ([NP] ution ) was calculated as the nanoparticle concentration ([NP] ideal The concentration of nanoparticles in the solution ([NP] 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 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 concentration of the gold salt used during synthesis, where it is assumed that all of it is reduced to gold. The mass per gold particle is obtained by approximating the NPs as spheres with a diameter corresponding to the peak of the size distribution (see Figure 3) and assuming that the density of gold NPs is the same as bulk gold. The number of MCs per unit volume comes from the manufacturer's specification of the number of MCs per gram of dry mass, which is 6.8 × 10 for Cytodex™-1. 6 g -1 and the mass of the dry powder was used.

[0042] Cytodex™-1 MC coated with AuNPs is easily distinguished from uncoated Cytodex-1, since bare MC is transparent in water and coated MC appears purple with an intensity that depends on the nanoparticle coverage (Figure 7a). The absorbance spectra of microcarriers coated with Au nanoparticles show a systematic increase in the peak intensity of the localized surface plasmon resonance band of the gold particles as a function of nanoparticle coverage (Figures 7b and c). The increase in peak intensity is also associated with a red shift of the peak wavelength (Figures 7b and c). Both changes reach a plateau at a saturation coverage of gold particles (Figure 7c). The red shift in the peak position is consistent with the expectation for plasmon coupling between neighboring particles, the chance of which increases with particle coverage. Saturation coverage occurs at nanoparticle concentrations of 3-fold or more [NP]. ideal This was achieved when the UV-Vis spectrum of the microcarriers was acquired using a TECAN Infinite M1000 PRO plate reader and recorded in the range of 250-1000 nm. A Greiner CELLSTAR™ 96-well plate was used as the plate reader. Samples were prepared as follows: 200 μl of each MC suspension (C0-C9) was added to a well of a microplate. The table below shows the reagents used for the functionalization of the microcarriers with AuNPs, namely the mass of Cytodex-1, the volume of the AuNP suspension (prepared as shown above) and the volume of water added each time to a total volume of 2 mL.

[0043] [Table 1]

[0044] Various masses of commercial MC powder were weighed directly into 9 different Eppendorf tubes. The respective volumes of AuNP suspension and water were then added. The MC was incubated for 3 hours. After incubation, the MC had settled and the supernatant was removed. The MC was then Adding water to the Eppendorf tube and pipetting up and down to wash all the MC out of the solution; Centrifugation at 1300 g for 2 minutes; and Wash three times by removing the supernatant.

[0045] The AuNP-coated MCs were then characterized with respect to their spectra.

[0046] The inelastic scattering properties were evaluated by Raman spectroscopy. A Renishaw inVia™ Raman microscope equipped with a high power near infrared laser diode operating at 785 nm and a visible laser diode operating at 633 nm was used to measure the intensity of the inelastic scattering from 100 to 4000 cm -1 Raman scattering spectra were recorded over a wide range of 100 nm to 100 nm. Before acquiring the spectra, the laser light was focused using an optical microscope (Olympus, objective, ×50L). The laser power was 0.9 mW (10%). For each spectrum, six 5 second accumulations were recorded. To ensure a representative characterization of the surface, multiple measurements were performed both at different sites of the same microcarrier and on different microcarriers. The results were analyzed using WiRE™ and OriginLab™ software. The acquired spectra were smoothed and normalized. The samples were prepared as follows: some MC was placed on a carbon tab.

[0047] Cell culture studies were performed using AuNP-coated MC (Cytodex™-1) and its uncoated version. As shown in Figure 8, cell kinetic growth was monitored by daily measuring the time evolution of glucose and lactate concentrations. Glucose is the main source of energy for cells, and lactate is one of the metabolic by-products of glucose metabolism. Thus, glucose and lactate concentrations are important indicators of cell growth and metabolic activity. In cell culture fermentation, about 50-100% of glucose is usually converted to lactate. Cells on AuNP-coated Cytodex™-1 MC consumed glucose similarly to cells on uncoated Cytodex™-1 MC. The rate of glucose consumption slowed when glucose was limiting, and as a result, the rate of lactate production decreased. No significant differences were observed between commercial Cytodex™-1 and AuNP-coated Cytodex™-1 MC.

[0048] Prior to cell culture, Cytodex™-1 MC should be swollen in PBS (phosphate buffered saline) and autoclaved at 120°C to sterilize them. However, Cytodex-1 MC was not autoclaved prior to gold nanoparticle attachment. After AuNP attachment, cell culture was performed on both autoclaved and non-autoclaved AuNP-coated Cytodex™-1, as this may affect the interaction of gold nanoparticles with the microcarriers.

[0049] Quantification of cell viability was performed by fluorescence microscopy. 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. Cells were found to colonize AuNP-coated Cytodex™-1 more extensively when they were not autoclaved. Therefore, autoclaving has an effect on the microcarriers that inhibits cell adhesion. However, the interaction between AuNP and Cytodex™-1 MC did not seem to be affected, i.e., the microcarriers remained opaque after autoclaving, which confirmed the presence of AuNP. Without wishing to be bound by theory, the inventors suggested that one reason for the decreased cell adhesion could be that the electrostatic interaction between gold nanoparticles and citrate (a stabilizer for gold nanoparticles) was broken by the high temperature. Removal of the negatively charged citrate molecules will change the overall charge on the surface of the microcarriers, thereby affecting their interaction with cells.

[0050] We further tested whether cells cultured on commercially available microcarriers would migrate to microcarriers dotted with plasmonic nanoparticles. The migration assay involved adding (non-autoclaved) AuNP-coated Cytodex™-1 to cell cultures on bare but otherwise identical Cytodex™-1 microcarriers.

[0051] 2 mL of Cytodex™-1 suspension (20 g / l) and 25 mL of cell culture medium were added to a 125 mL Erlenmeyer flask. The MCs were kept in the vessel at 37° C. and 5% CO2 for 1 h. The cells were detached from their vessel flasks by trypsinization and harvested. They were added to the Erlenmeyer already containing the microcarriers (0.8×10 5Cells were incubated in cell culture medium to allow the cells to adhere onto the MCs. After 1 hour, the microcarrier-tethered cells were stirred at 70 RPM (37°C, 5% CO2). Samples of cell culture medium were withdrawn daily to measure glucose and glutamine consumption and 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 and dead, respectively.

[0052] On day 4, 50% of the cell culture medium was replaced. On day 4, gold nanoparticle-coated microcarriers were added to the Cytodex™-1 culture flask. After 1 hour of incubation, agitation was resumed (70 RPM). Quantification of cells was performed daily by fluorescence microscopy. Cells were mixed with DAPI to stain the cells. The cells were able to migrate towards the new carriers, confirming the ability of the modified microcarriers to support cell proliferation function.

[0053] In summary, the optical properties of microcarriers coated with plasmonic nanoparticles correlate with the coverage of gold nanoparticles and show strong plasmonic coupling at saturation coverage. Plasmonic NP-coated MCs open the possibility of detecting smaller organic molecules using SERS or other plasmonic sensing techniques. Comparing plasmonic NP-coated MCs with their uncoated counterparts showed similar cell growth kinetics and migration of human mesenchymal stem cells, providing the first evidence for the retention of cell proliferation functionality. The plasmonic microcarriers reported here pave the way for future plasmonic sensing of changes at the interface between the microcarrier and biological medium and for monitoring changes to biological cells in different phases of cell growth and potentially enabling plasmonic liberation of cells by plasmonic heating action.

[0054] Although specific embodiments have been described in detail herein, those skilled in the art will appreciate that various modifications and alternatives to those details may be developed in light of the overall teachings of the present disclosure. Accordingly, the specific arrangements disclosed are intended to be illustrative only and not limiting as to the scope of the invention which is to be given the scope of the appended claims and any and all equivalents thereof. [Explanation of symbols]

[0055] 10 Microcarrier beads 12 Beads 14 Plasmonic Nanoparticles 16 cells 112 Microcarriers 114 Gold Nanoparticles

Claims

1. A cell culture reactor containing a cell culture medium and microcarrier beads for cell culture, A cell culture reactor, comprising microcarrier beads for cell culture, the microcarrier beads being made of a bead body having a surface dotted with plasmonic nanoparticles, the bead body having a diameter in the range of 50 μm to 1 mm, and the bead body comprising a swollen or non-swollen hydrogel.

2. 2. The cell culture reactor of claim 1, wherein the plasmonic nanoparticles comprise plasmonic metal nanoparticles.

3. 2. The cell culture reactor of claim 1, wherein the plasmonic nanoparticles comprise gold nanoparticles.

4. 4. The cell culture reactor of claim 1, wherein the plasmonic nanoparticles are stabilized with a capping agent.

5. The cell culture reactor of claim 1 , wherein the plasmonic nanoparticles are spherical.

6. The cell culture reactor of any one of claims 1 to 5, wherein the plasmonic nanoparticles have a diameter in the range of 2 to 200 nm.

7. 7. The cell culture reactor according to claim 1, wherein the ratio of the diameter of the bead bodies to the diameter of the plasmonic nanoparticles is in the range of 250 to 25,000.

8. A microcarrier bead for cell culture, comprising a bead body having a surface dotted with plasmonic nanoparticles, said bead body having a diameter in the range of 50 μm to 1 mm, said bead body comprising a swollen or non-swollen hydrogel, and said plasmonic nanoparticles being stabilized with a capping agent.

9. 1. A method for observing live cells on microcarrier beads, the microcarrier beads being made of bead bodies having a surface dotted with plasmonic nanoparticles, the bead bodies having a diameter in the range of 50 μm to 1 mm, the bead bodies comprising a swollen or non-swollen hydrogel, comprising: illuminating the microcarrier beads with a probe light to excite localized surface plasmons in the plasmonic nanoparticles; detecting light emitted from the plasmonic nanoparticles in response to illumination. method.

10. 10. The method for observing living cells according to 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. 1. A method for culturing living cells on microcarrier beads, the method comprising: culturing live cells on microcarrier beads, the microcarrier beads being made of bead bodies having a surface dotted with plasmonic nanoparticles, the bead bodies having a diameter in the range of 50 μm to 1 mm, the bead bodies comprising a swollen or non-swollen hydrogel, the method comprising: The method of claim 1, wherein release of cells from said microcarrier beads is stimulated by exciting localized surface plasmon resonance in said plasmonic nanoparticles.

12. The method of claim 11 , wherein the cell liberation is stimulated by plasmonic heating of the nanoparticles.

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