WO2018187840A1

WO2018187840A1 - Method and substrate for cell recovery

Info

Publication number: WO2018187840A1
Application number: PCT/AU2018/050328
Authority: WO
Inventors: Yashaswini VEGI; Simon Moulton; Nicholas Reynolds
Original assignee: Swinburne University Of Technology
Priority date: 2017-04-13
Filing date: 2018-04-11
Publication date: 2018-10-18

Abstract

The present invention relates to cell culture and the use of nanorods comprising a plasmonic metamaterial for facilitating detachment of cells from the cell culture. These substrates are optically responsive and capable of stimulating plasmons which are capable of detaching cells from the cell culture. Cell populations retrieved from the cell cultures are also provided.

Description

METHOD AND SUBSTRATE FOR CELL RECOVERY

FIELD OF THE INVENTION

The present invention relates to cell culture substrates and the use of nanorods comprising a plasmonic metamaterial for facilitating detachment of cells from a cell culture. These substrates are optically responsive and capable of stimulating a surface plasmon resonance which is capable of detaching cells from the cell culture. Cell populations retrieved from the cell cultures are also provided. BACKGROUND OF THE INVENTION

Targeted detachment of cells and cell harvesting from cell cultures is of significant interest in biomedical research, regenerative medicine, and tissue engineering. A lack of inexpensive, simple, specific and mild cell recovery methods is one of the factors impeding the development of methods to culture clinically relevant numbers of therapeutic stem cells.

Enzymatic cell detachment is the industry standard technique for therapeutic cell recovery. This standard procedure for detaching cells from a culture substrate includes the use of digesting enzymes and often animal derived enzymes which may irreversibly damage cells. The use of animal products can also be undesirable depending on the final use of the cells. Trypsinization and other detachment methods including ethylenediaminetetraacetic acid (EDTA), trypsin, Collagenase, or Dispase have many short comings including being derived from animal/human pancreatic enzymes, considerable expense, and the cleaving of cell surface proteins leading to dysregulation of cell function, inducing apoptosis in cells when exposed for longer time periods. There are a number of non-enzymatic solutions for cellular detachment available; however these methods show only moderate efficiency and are generally unsuited for scale up to clinically relevant volumes. Additionally, physical methods of scraping the cells from a culture plate can cause harm to the cells such that they are damaged and require repair before they are capable of further culturing. Furthermore, this process is not scalable for large volumes of cells. A minimally invasive and efficient method for detaching cells or a method that avoids the use of digesting enzymes is desired.

Some methods involve light irradiation of a defined surface region or application of direct UV- or near-infrared (NIR) radiation for targeted cell detachment. However, some of these methods have limitations on the cell culture surface, media or may affect biological molecules or may be toxic to cells.

These methods use nanoparticles in cell culture which have shown that they may reduce the damage to cells during the detachment process. These nanoparticles are used as plasmonic substrates which show a broad plasmon band covering a wide part of the visible and near-infrared (NIR) spectral ranges. Subsequent irradiation with a NIR laser results in plasmon stimulation and highly efficient detachment of the cells. Generally, NIR is safer and more efficient (compared to visible light) as it will not be absorbed by biomolecules in the media, which may lead to loss of intensity but also unwanted macroscale photo-stimulation of the cells.

Remote irradiation with light stands out as one of the most convenient triggers for cell detachment. Visible green light has been used to induce cell detachment from a substrate covered with small gold nanoparticles. However these light sources can significantly damage biological material.

Surface-plasmons generated by irradiating surface bound nanostructures coated with plasmonic materials with near infra-red light can mediate non-toxic cell detachment. Plasmons are stimulated by the combined use of irradiation and these optically responsive surfaces. Light in the near-infrared (NIR) range (800-2500 nm) appears as a highly convenient stimulus for cell detachment as it is biocompatible, displays significant transmission in biological fluids and tissues, minimal heating of aqueous media, and most importantly, no induced photomodification of biomolecules.

On the surface of many conducting materials (most commonly noble metals such as gold and silver), there are a large numbers of surface bound delocalised electrons not associated with specific atoms. This 'sea' of delocalised electrons (also known as a surface plasmon) can adsorb electromagnetic radiation (i.e. photons from an IR laser) exciting the surface plasmons and producing standing waves of varying electron density that spread across the conducting surface (surface plasmon resonance). The conversion of incident radiation to excited surface plasmons is very sensitive to adsorbed materials on the conducting surface.

Unique plasmonic effects occur due to the amplitude of the light wave being increased by an order of magnitude due to photon confinement. Due to the fact that the intensity of light is directly proportional to the square of the wave's amplitude there is an increase in the intensity of light. In this manner, noble metal nanoparticles, through plasmonic confinement, effectively focus resonantly coupled light. Due to this there is an enhancement in the radiative properties of the metal (fluorescence, Rayleigh scattering, Raman spectroscopy, localised heating etc.). The smaller size of the nanoparticles can greatly increase the optical resolution. This phenomenon is due to the strong surface electromagnetic field generated around the nanoparticle and its exponential decay over distances similar to the particle size. The strong electromagnetic field is caused by the resonant photons which are confined within the plasmonic nanoparticles and causes the local surface plasmon oscillation. For these reasons the energy produced from the surface plasmon resonance of nanoparticles can be harnessed and used in a variety of applications. The above step if followed by rapid dephasing of the electrons which is in sync with the equally rapid transfer of energy to the lattice. This process is essential in technologies which are based on the photothermal properties related to the plasmonic nanoparticles.

In 2012, Kolesnikova, T.A., et al., demonstrated that the selective detachment of cells by using gold nanoparticles was possible. Micro contact printing to selectively deposit gold nanoparticles in specific micro patterns was used. Cell detachment was achieved by exposing NIH3T3 cells grown on gold nanoparticles base to visible green laser light (520nm). This may have led to the nanoplasmonic effect which may have triggered a photothermal or photochemical response leading to cell detachment.

In 2016, Giner-Casares, J. I., et al., executed a set of experiments where a plasmonic substrate was created by depositing the gold nanoparticle "seeds". A dense array of gold nanostructures was grown on the surface by the process of chemical growth. Five different cells (HeLa, A549, HUVECS, T3T and J774) were used to test the detachment process. The cell detachment for each of these cells was successful when the surface was irradiated with NIR of 980 nm. It was observed that each different cell line required different exposure times. HUVECS took the least time (5 minutes), J775 and A594 took the highest time to detach (40 minutes), with detachment rate ranging from 80% - 100%. The viability of the detached cells valued between 75 - 100%. When the detached cells were re-seeded for culture, relevant amounts of adhesion and proliferation was observed.

However, despite the effective use of gold nanoparticles with NIR, the consistent size of the nanoparticles provides little manoeuvrability with the stimulus (laser) that provides the light source to stimulate plasmons. Additionally the chemical growth process used to generate the nanostructures used was complex and requires significant experimental expertise limiting the applications of such complex nanostructures. The use of visible green light with gold nanoparticles can still damage cells and biomolecules whereas the use of NIR with the gold nanoparticles requires complex fabrication processes.

Hence, it would be desirable to provide a method or substrate or cell culture that can effectively detach cells and which is convenient to use and efficient to scale up for large volumes of cells whilst minimalizing damage to cells and reducing impact on the cells and biological components during the detachment process.

SUMMARY OF THE INVENTION

In one aspect of the present invention there is provided a cell culture substrate coated with nanorods (NR) comprising a plasmonic metamaterial. Preferably the nanorods are gold nanorods. More preferably, the nanorods have a high aspect ratio and enable tunability to a desired wavelength for tuning a surface plasmon.

In another aspect of the present invention there is provided a cell culture plate comprising the cell culture substrate coated with NR comprising a plasmonic metamaterial.

In yet another aspect of the present invention, there is provided a method for cell detachment from a cell culture substrate said method comprising culturing cells on a cell culture substrate coated with NR comprising a plasmonic metamaterial for a period of time sufficient for the cells to attach to the NR and optionally for the cells to proliferate on the NR; and

stimulating a surface plasmon on the NR for a time sufficient to detach the cells from the NR; and

optionally recovering the cells from the cell culture.

In yet another aspect of the present invention there is provided a method for preparing a cell culture substrate for cell culture said method comprising

selecting a NR comprising a plasmonic metamaterial with an aspect ratio enabling tunability to a desired wavelength;

preparing a substrate for integration of the NR on a surface of the cell culture substrate or embedding into the cell culture substrate; and

integrating or embedding the substrate with the NR such that the NR are accessible to cells for attachment and culture.

In another aspect there is provided a NR comprising a plasmonic metamaterial when used for cell culture and detachment of cells from the cell culture. Preferably the nanorods are gold nanorods. More preferably, the nanorods have a high aspect ratio and enable tunability to a desired wavelength for tuning a surface plasmon.

In another aspect of the invention, there is provided a cell population comprising cells optionally recovered from a method according to the present invention. Other aspects of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the invention.

Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification (including the claims) they are to be interpreted as specifying the presence of the stated features, integers, steps or components, but not precluding the presence of one or more other features, integers, steps or components, or group thereof. DESCRIPTION OF THE FIGURES

Figures 1 A and 1 B show (A) Protein A- N-hydroxysuccinimide functionalized gold nanorods coated surface or (B) Poly Amine functionalized gold nanorods coated surface taken under scanning electron microscope.

Figure 2 shows an illustration of the experimental set up.

Figure 3 shows the cell detachment process.

Figure 4(A) and 4(B) show NIH-3T3 cells cultured on Poly Amine coated gold nanorods on a silicon oxide substrate surface before (A))and after (B)exposure to near infra-red laser taken by an Inverted microscope. Figure 5(A) and 5(B) show MSC cells cultured on Poly Amine coated gold nanorods on a silicon oxide substrate surface before (A) and after (B)exposure to near infra-red laser taken by an Inverted microscope.

DETAILED DESCRIPTION OF THE INVENTION

Detachment of viable cells from a cell culture substrate is paramount to successful cell culture and continued propagation of cell lines for areas of significant interest such as but not limited to biomedical research, regenerative medicine, and tissue engineering. The ability to effectively retrieve cells with minimal disruption means that the cells are in better condition, remain intact and often are more responsive to propagation and expansion.

In one aspect of the present invention there is provided a cell culture substrate for culturing cells said substrate coated with nanorods (NR) comprising a plasmonic metamaterial.

In one embodiment the cell culture substrate of the present invention may be glass, plastic, metal, surface treated glass, surface treated plastic, surface treated metal, silicon, polymeric material or any other material that can receive NR and which are non-toxic to cells. Additionally, it is preferred that the substrate is not light sensitive; in particular, is preferably inert and does not degrade or react to light. Preferably the substrate is silicon or silicon dioxide.

The substrate must be prepared such that it is suitable for nanorod deposition and as well as for cell attachment and culture. Preparation may include use of UV/ozone.

The substrate may be 2-Dimensional or 3-Dimensional such as but not limited to any types of surface for cell culture including cell culture plates, beads, scaffolds, flasks and culture sheets.

In one embodiment the NR are functionalized to facilitate cell attachment. This means that the NR have added functional groups, biomolecules or ligands that enable cells to attach. Suitable functional groups, biomolecules or ligands may be selected from the group comprising, but not limited to, biological molecules, inorganic materials, synthetic polymers, natural polymers and monomers such as nucleic acids, proteins and peptides, carbohydrates, antibodies, growth factors, cytokines, receptors, polypeptides, lipids, or steroids and molecules such as biotin, avidin, neutravidin, streptavidin, or biotinylated antibodies, and ligands. The most suitable functionalization will depend on the cell type that is to be cultured and the conditions of culture. A person skilled in the art would be capable of determining suitable functionalization to enhance cell culture for the cell type involved. For instance, culturing NIH 3T3 cells and MSC, polyamine coated NR are preferred.

In yet another embodiment the NR have a high aspect ratio enabling tunability to a desired wavelength for tuning a surface plasmon. Nanorods typically have high aspect ratios, whereas nanoparticles or nanospheres are generally spherical and have a low aspect ratio.

An aspect ratio describes a proportional relationship between width and height (or length). Therefore a high aspect ratio will have a greater length to width and hence will be long rather than spherical which would have a low aspect ratio.

The nanorods with a high aspect ratio have the advantage when it comes to tunability. By changing the dimensions of the nanorods even slightly the surface plasmon resonance can be tuned to a desired wavelength. Optimal aspect ratio may be selected so that a surface plasmon resonance exists in a desired range such as the NIR range. This enables a stimulus (a laser) to be used that does not undergo significant absorption in biological media, unlike with the spherical nanoparticles which use a green laser, which will undergo significant adsorption in biological media.

For gold nanorods, tunability can be defined as the change in length or diameter of the GNR in order to get a preferred aspect ratio. With the change in aspect ratio, the surface plasmon resonance peak (SPR peak) of the GNR changes. For example, while gold nanorods with an aspect ratio of 2.9 (1 Onm in diameter and 29nm in length) has a SPR peak at 700nm, gold nanorods with an aspect ratio of 5.9 (10nm in diameter and 59nm in length) will have its SPR at 980nm.

The aspect ratio may vary depending on the wavelength so desired. Preferably, an aspect ratio in the range of 1 .9 - 8.1 may be selected for 600nm-1200nm. More preferably, the aspect ratio is in the range of 2.9 to 5.9 for wavelengths from 700nm - 980nm. For a wavelength at 780nm the preferred aspect ratio for surface plasmons to react is in the range of 3.5 to 3.8, preferably 3.5, or 3.6, or 3.7, or 3.8 However, in one embodiment, it is preferred that the aspect ratio is selected to enable tuning a surface plasmon resonance in the range of 600nm to 1200nm. More preferably the aspect ratio is selected to enable tuning a surface plasmon resonance in the range of 700nm - 1000 nm, or 700nm - 750nm, or 750 nm - 1000 nm, or 750 nm - 950 nm, or 750 nm - 900 nm, or 750 nm - 850 nm, or 750 nm - 800 nm, or the wavelength is selected from 755 nm, or 760 nm, or 765 nm, or 770 nm, or 775 nm, or 780 nm, or 785 nm, or 790 nm, or 795 nm, or 800 nm, preferably the wavelength is selected from 780 nm, or 781 nm, or 782 nm, or 783 nm, or 784 nm, or 785 nm, or 786 nm, or 787 nm, or 788 nm, or 789 nm or 790 nm. Most preferably the wavelength is 785nm.

In another embodiment the NR are integrated on a surface of the cell culture substrate or embedded into the cell culture substrate providing access to cell attachment. When preparing the substrate or an optically responsive substrate for cell culture, the NR should be accessible to the cells, at least so that the cells can attach and be detached by stimulating a plasmon by exposure to a laser. Generally, the NR are coated on the surface of the substrate. Where they may be embedded into the substrate, there must be exposure of NR for attachment of cells. NR may be coated onto a surface by simply pipetting a suspension of NR onto the surface or substrate at a required density to evenly spread over the surface and allowed to dry. Excess NR may be washed off prior to cell culture.

In one embodiment the substrate comprises the NR at a density in the range of 10 - 1000 NR/μιη². However, the density can vary. For example, in the case of Protein A functionalized NR, the cells may detach when the density is between 10 - 50 NR/μιτι². In the case of Poly Amine functionalized NR, the density may be between 300 - 400 NR/μιη². However, the density of NR may be optimized so that a uniform layer is obtained. Preferably, the density is in the range of 10 - 500 NR/μιτι² 10 - 400 NR/μιη²' 10 - 300 NR/μηπ², 10 - 200 NR/μηπ², 10 - 100 NR/μηπ² or 10 - 50 NR/μηπ².

The NR comprise a plasmonic metamaterial. Plasmonic metamaterial have a special advantage to control electromagnetic wave propagation and especially adjust the light polarization state through different designs or diverse spatial arrangements of a structural unit. Plasmonic metamaterial may be selected from the group including gold, silver, copper, titanium, titanium dioxide, graphene, silicon, and germanium. Preferably, the plasmonic metamaterial is not toxic to the cells but can stimulate a plasmon when exposed to a laser and preferably at a wavelength that is desirable to not cause damage to the cells or surrounding biological materials. Most preferably, the plasmonic metamaterial is gold or the NR are gold NR.

In another aspect of the present invention there is provided a cell culture plate comprising the cell culture substrate as herein described. The cell culture plate may comprise the substrate integrated in the material of the cell culture plate, or it may include a cell culture sheet that comprises the substrate with the NR which is then placed within the cell culture plate. For instance, for flasks or single culture plates or wells, a culture sheet may be placed into the flask or single culture well and be removable so that the remaining culture plate or well can be reusable.

In yet another aspect of the present invention, there is provided a method for cell detachment from a cell culture substrate said method comprising

culturing cells on a cell culture substrate coated with NR comprising a plasmonic metamaterial for a period of time sufficient for the cells to attach to the NR and optionally for the cells to proliferate on the NR; and

optionally recovering the cells from the cell culture.

The cell culture substrate as herein described can be used as part of a cell culture system such that when the cells are cultured can facilitate the detachment of the cells for retrieval either for further passage or experimentation.

In the method of the present invention, the cells are cultured on the cell culture substrate as herein described. Cells are introduced to the NR comprising a plasmonic metamaterial that are integrated with the substrate or coated on the substrate and allowed to attach. Preferably the cells will be allowed to grow for a period of time that allows them to attach and preferably proliferate. This will depend on the cell type in culture. Any cell type may be cultured using the cell culture substrate of the present invention. Suitable cell types will include but are not limited to fibroblasts, stem cells such as mesenchymal stem cells, haematopoietic stem cells, embryonic stem cells, cardiomyocytes, kidney cells, liver cells etc. A person skilled in the art will adapt the cell type to the culture conditions.

Once the cells are attached, they may be detached by stimulating a surface plasmon resonance.

Surface plasmons can be stimulated using a terahertz device and visible wavelength.

Preferably the wavelength for stimulating the surface plasmon is in the range of 600nm - 1200nm, preferably the wavelength is in the range of 700nm - 1000 nm, or 700nm - 750nm, or 750 nm - 1000 nm, or 750 nm - 950 nm, or 750 nm - 900 nm, or 750 nm - 850 nm, or 750 nm - 800 nm, or the wavelength is selected from 755 nm, or 760 nm, or 765 nm, or 770 nm, or 775 nm, or 780 nm, or 785 nm, or 790 nm, or 795 nm, or 800 nm, preferably the wavelength is selected from 780 nm, or 781 nm, or 782 nm, or 783 nm, or 784 nm, or 785 nm, or 786 nm, or 787 nm, or 788 nm, or 789 nm or 790 nm. Most preferably the wavelength is 785nm.

For gold nanorods NIR is particularly effective as the aspect ratios in the range of 1 .9 - 8.1 at a wavelength between 600nm - 1200nm is preferable. An NIR laser may be used.

The time sufficient to detach the cells from the substrate will depend on the cell type. The time period of exposure changes for different cell types. Studies show that while some cells take only 5 minutes others may take up to 30 to 40 minutes. Applicants have followed a standard exposure time period of 1 hour. Fibroblasts are said to be the most adhesive cells and applicants have found that they detach easily after one hour of exposure. Therefore, to determine whether the cells have detached, the cell culture surfaces can be easily viewed under a microscope. If further time is needed, the substrate may be further exposed to the laser. Generally, the laser is applied as a continuous wave.

preparing a substrate for coating of the NR on a surface of the cell culture substrate or embedding into the cell culture substrate; and

coating or embedding the substrate with the NR such that the NR are accessible to cells for attachment and culture.

The cell culture substrates of the present invention can be prepared by providing a source of NR comprising a desired plasmonic metamaterial selected from but not limited to gold, silver, copper, titanium, titanium dioxide, graphene, silicon, and germanium. Preferably the NR are gold NR (GNR). NR may be obtained commercially. One commercial source is from Nanopartz™ (http://www.nanopartz.com/). The NR may be further functionalized as herein described by methods available to the skilled addressee and selected to have a high aspect ratio enabling tunability to a desired wavelength for tuning a surface plasmon as herein described. Alternatively NR may be purchased already functionalized for the cell type to be cultured. Before using the NR, they are preferably treated to remove any potential aggregation between the nanorods. This may be conducted in an ultrasonic bath for approximately 7 - 10 minutes.

The NR may be suspended in phosphate buffered saline (PBS) and diluted to the required density. An amount of the diluted NR solution may be pipetted onto the substrate and evenly spread for integration with the substrate. The surfaces may be dried overnight allowing the NR to settle down and attach to the substrate. The following day the surfaces may be washed with PBS to remove any excess or unattached NR.

Prior to adding the NR to the substrate, the surface of the substrate may be prepared and cleaned such as by exposure to UV/ozone cleaner. The exposure may last for a time period of approximately one hour or for a period sufficient to clean the surface. The substrates comprising the NR must be sterilized prior to use for cell culture. Any methods available to sterilize the surface may be used providing the method does not damage the interactions with the NR and the substrate. However, one option is by exposing them to 2% Anti-Anti (Antimycotic-Antibiotic, GIBCO) solution for at least 60 minutes and then washed and rinsed with PBS.

Cells may now be seeded on the substrates at a required density and incubated for 24 hours, allowing them to attach and proliferate. In a further embodiment of the invention there is provided a cell culture substrate for cell culture prepared by the methods described herein.

In another aspect there is provided a NR comprising a plasmonic metamaterial when used for cell culture and detachment of cells from the cell culture.

Prior attempts to use nanoparticles for cell detachment utilized gold nanostructures to promote the detachment of cells. The nanoparticles were generally nanospheres have low aspect ratios and thereby requiring wavelengths such as visible green light at 520nm more likely to be absorbed by biomolecules in the media, which may have lead to loss of intensity but also unwanted macroscale photo-stimulation of the cells.

Where NIR has been used, the nanostructures were as a result of complex fabrication procedures to generate complex nanostructures that are capable of generating a surface plasmon resonance in the NIR. However, these complex structures failed to provide the versatility that is provided by NR. Due to the availability of NR with a range of easily accessible aspect ratios NR of the present invention have greater tunability enabling variability of the surface plasmon resonance at a desired wavelength. Selecting an aspect ratio so that the surface plasmon resonance exists in the NIR range enables utilization of a stimulus (the laser) that does not undergo significant absorption in biological media, unlike with the spherical nanoparticles which use a green laser, which will undergo significant adsorption in biological media.

In this aspect of the invention the NR are functionalized as herein described to facilitate cell attachment. They have a high aspect ratio enabling tunability to a desired wavelength for tuning a surface plasmon. Preferably, the aspect ratio enables tuning a surface plasmon resonance in the range of 600nm to 1200nm, preferably the wavelength is in the range of 700nm - 1000 nm, or 700nm - 750nm, or 750 nm - 1000 nm, or 750 nm - 950 nm, or 750 nm - 900 nm, or 750 nm - 850 nm, or 750 nm - 800 nm, or the wavelength is selected from 755 nm, or 760 nm, or 765 nm, or 770 nm, or 775 nm, or 780 nm, or 785 nm, or 790 nm, or 795 nm, or 800 nm, preferably the wavelength is selected from 780 nm, or 781 nm, or 782 nm, or 783 nm, or 784 nm, or 785 nm, or 786 nm, or 787 nm, or 788 nm, or 789 nm or 790 nm. Most preferably the wavelength is 785nm. In this aspect of the present invention the nanorods with a high aspect ratio and can be tuned to a desired wavelength. An optimal aspect ratio may be selected so that a surface plasmon resonance exists in a desired range such as the NIR range. Preferably the range enables a stimulus (a laser) to be used that does not undergo significant absorption in biological media, unlike with the spherical nanoparticles which use a green laser, which will undergo significant adsorption in biological media.

For gold nanorods (GNR), tunability can be defined as the change in length or diameter of the GNR in order to get a preferred aspect ratio. With the change in aspect ratio, the surface plasmon resonance peak (SPR peak) of the GNR changes. For example, while gold nanorods in this aspect with an aspect ratio of 2.9 (10nm in diameter and 29nm in length) has a SPR peak at 700nm, gold nanorods with an aspect ratio of 5.9 (10nm in diameter and 59nm in length) will have its SPR at 980nm.

The aspect ratio may vary depending on the wavelength so desired. Preferably, an aspect ratio of betweenin the range of 1 .9 - 8.1 may be selected for 600nm-1200nm. More preferably, the aspect ratio is of in the range of 2.9 to 5.9 for wavelengths from 700nm - 980nm. For a wavelength at 780nm the preferred aspect ratio for surface plasmons to react is in the range of 3.5 to 3.8, preferably 3.5, or 3.6, or 3.7, or 3.8.

In this aspect of the invention the NR may comprise a plasmonic metamaterial selected from gold, silver, copper, titanium, titanium dioxide, graphene, silicon, and germanium. Preferably the NR are gold NR (GNR).

In another aspect of the invention, there is provided a cell population comprising cells optionally recovered from a method according to the present invention.

Since the method of detachment employed by the methods of the present invention are gentle and less likely the harm the cells, the cell population includes cells that are unharmed and can be more effectively re-cultured. They tend to be more robust and have a greater potential increase in differentiation ability. The present invention will now be more fully described by reference to the following non-limiting Examples.

EXAMPLES

Example 1 : Preparation of a Cell Culture Substrate comprising Gold Nanorods (GNR) for Cell Culture

(i) Preparation of a silica surface

A silica surface such as a glass surface and silicon wafers was prepared and exposed to UV/ozone cleaner for 1 hour to clean the surface prior to application of gold nanorods.

(ii) Preparation of GNR

Functionalized gold nanorods (GNR) were obtained from Nanopartz (USA) (http://www.nanopartz.com/) as gold nanorod colloids. The GNR were functionalized with Protein A- N-hydroxysuccinimide (Figure 1 A) or Poly Amine (Figure 1 B). The gold nanorod colloids where put in an ultra sonicator bath for 7 minutes in order to remove any potential aggregation between the nanorods. PBS was added to the GNR colloid to dilute the GNR to a required density. 70-80μΙ of the diluted solution was pipetted on to the substrate and spread evenly to obtain a spread of GNR at a density in the range of 10 - 1000 NR/μιτι². The surfaces were allowed to dry overnight allowing the gold nanorods to settle down and attach to the material. The following day, the surfaces were washed to PBS to remove any excess or unattached gold nanorods. Figures 1 A and 1 B show (A) Protein A- N-hydroxysuccinimide functionalized gold nanorods coated surface or (B) Poly Amine functionalized gold nanorods coated surface. The white features observed in the picture of Figure 1 (A) are the GNR bound to the surface by the process of nonspecific absorption. It can also be seen that the gold nanorods tend to aggregate together. In Figure 1 (B) an even spreading of the gold nanorods on the surface is observed due to electrostatic repulsions between the polyamine coatings. The nanorods are bound to the surface by the process of non-specific absorption. (Hi) Preparation of a GNR-substrate for cell culture and cell culture: The sterilization of a GNR substrate was performed by exposing the substrate to 2% Anti-Anti (Antimycotic-Antibiotic, GIBCO) solution for at least 60 minutes. After 60 minutes, the substrates are thoroughly washed with PBS to remove excess Anti-Anti present on them. Cells (such as NIH 3T3 cells or Mesenchymal Stem Cells (MSC)) were seeded on to this surface at a required density and incubated for 24 hours, allowing them to attach and proliferate.

Example 2: Culturing and Detaching Cells from a Cell Culture Substrate comprising Gold Nanorods (GNR)

(i) Laser exposure

NIH 3T3 cells and MSC were allowed to grow on the cell culture substrate for 24 hours. After 24 hours of cell culture, the cells were exposed to a laser source of near infra-red laser (NIR) with continuous wavelength. The Wavelength was set at 785nm. The Fiber optic core was 200μιη and the Power density with the collimator was 800mW/cm². Figure 2 shows an illustration of the experimental set up.

The laser was set up at a height of 10mm±2 from the cell culture samples (all experiments were done on a 24 well plate). Each of the samples were exposed for a time period of one hour to stimulate plasmons and detach the cells. After one hour, the samples were washed gently to remove detached cells.

Figure 3 shows the cell detachment process. This illustrates a simple overview of the cell detachment process from the GNR coated silicon oxide surface. The first panel shows the attachment and proliferation of cells on the substrate. In the second panel the cells are exposed to near-infra red light. The spiked ovals on the gold nanorods indicates the surface plasmons reacting to the exposure of laser. The third panel shows that the cells have been released from the surface after being exposed to the laser for required period of time.

Figure 4(A) and 4(B) show NIH-3T3 cells cultured on Poly Amine coated gold nanorods silicon oxide substrate surface before (Figure 4(A)) and after (Figure 4(B) exposure to near infra-red laser taken by an scanning electron microscope. Figure 5(A) and 5(B) show MSC cells cultured on Poly Amine coated gold nanorods surface before (Figure 5(A)) and after (Figure 5(B) exposure to near infra-red laser taken by an scanning electron microscope. The detached cells were then re-seeded on to another culture plate for further observation for their continued viability, ability to proliferate upon reseeding and continued differentiation ability.

While the foregoing written description of the invention enables one of ordinary skill to make and use what is considered presently to be the best mode thereof, those of ordinary skill will understand and appreciate the existence of variations, combinations, and equivalents of the specific embodiment, method, and examples herein. The invention should therefore not be limited by the above described embodiment, method, and examples, but by all embodiments and methods within the scope and spirit of the invention as broadly described herein.

REFERENCES

1 . Kolesnikova, T.A., et al., Laser-Induced Cell Detachment, Patterning, and Regrowth on Gold Nanoparticle Functionalized Surfaces. Acs Nano, 2012. 6(1 1 ): p.

9585-9595.

2. Giner-Casares, J. I., et al., Plasmonic Surfaces for Cell Growth and Retrieval Triggered by Near-Infrared Light. Angewandte Chemie-lnternational Edition, 2016. 55(3): p. 974-978.

Claims

CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1 . A cell culture substrate for culturing cells said substrate coated with nanorods (NR) comprising a plasmonic metamaterial.

2. A cell culture substrate according to claim 1 wherein the NR are functionalized to facilitate cell attachment.

3. A cell culture substrate according to claim 1 or 2 wherein the NR have a high aspect ratio enabling tunability to a desired wavelength for tuning a surface plasmon.

4. A cell culture substrate according to claim 3 wherein the aspect ratio enables tuning a surface plasmon resonance in the range of 600nm to 1200nm.

5. A cell culture substrate according to any one of claims 1 to 4 wherein the NR are integrated on a surface of the cell culture substrate or embedded into the cell culture substrate providing access to cell attachment.

6. A cell culture substrate according to any one of claims 1 to 5 wherein substrate comprises the NR at a density in the range of 10 - 1000 NR/μιη².

7. A cell culture substrate according to any one of claims 1 to 6 wherein the NR comprise a plasmonic metamaterial selected from gold, silver, copper, titanium, titanium dioxide, graphene, silicon, and germanium.

8. A cell culture substrate according to any one of claims 1 to 7 wherein the plasmonic metamaterial is gold.

9. A cell culture plate comprising the cell culture substrate according to any one of claims 1 to 8.

10. A method for cell detachment from a cell culture substrate said method comprising

culturing cells on a cell culture substrate coated with NR comprising a plasmonic metamaterial for a period of time sufficient for the cells to attach to the NR and optionally for the cells to proliferate on the NR; and stimulating a surface plasmon on the NR for a time sufficient to detach the cells from the NR; and

optionally recovering the cells from the cell culture.

1 1 . A method according to claim 10 wherein the NR are functionalized to facilitate attachment of the cells to the NR.

12. A method according to claim 10 or 1 1 wherein the NR have a high aspect ratio enabling tunability to a desired wavelength for tuning a surface plasmon.

13. A method according to claim 1 2 wherein the aspect ratio enables tuning a surface plasmon resonance in the range of 600nm to 1200nm.

14. A method according to any one of claims 10 to 13 wherein the NR are integrated on a surface of the cell culture substrate or embedded into the cell culture substrate providing access to cell attachment.

15. A method according to any one of claims 10 to 14 wherein the substrate is coated with NR at a density in the range of 10 - 1000 GNR/μιτι²

16. A method according to any one of claims 10 to 15 wherein the surface plasmon is optically stimulated on the NR.

17. A method according to any one of claims 10 to 16 wherein the surface plasmon is stimulated by exposure to near infra-red (NIR) light.

18. A method according to any one of claims 10 to 17 wherein the surface plasmon is stimulated by exposure to near infra-red (NIR) light in the range of 600nm to 1200nm.

19. A method according to any one of claims 10 to 18 wherein the NR are coated with a plasmonic metamaterial selected from gold, silver, copper, titanium, titanium dioxide, graphene, silicon, and germanium.

20. A method according to any one of claims 10 to 19 wherein the plasmonic metamaterial is gold.

21 . A method for preparing a cell culture substrate for cell culture said method comprising

22. A method according to claim 21 wherein the NR are functionalized to facilitate cell attachment.

23. A method according to claim 21 or 22 wherein the NR have a high aspect ratio enabling tunability to a desired wavelength for tuning a surface plasmon.

24. A method according to any one of claims 21 to 23 wherein the aspect ratio of the NR enables tuning a surface plasmon resonance in the range of 600nm to 1200nm.

25. A method according to any one of claims 21 to 24 wherein the NR are integrated on a surface of the cell culture substrate or embedded into the cell culture substrate providing access to cell attachment.

26. A method according to any one of claims 21 to 25 wherein the NR are coated at a density in the range of 10 - 1000 NR/μιτι².

27. A method according to any one of claims 21 to 26 wherein the NR comprise a plasmonic metamaterial selected from gold, silver, copper, titanium, titanium dioxide, graphene, silicon, and germanium.

28. A method according to any one of claims 21 to 27 wherein the plasmonic metamaterial is gold.

29. A cell culture substrate for cell culture prepared by the method according to any one of claims 21 to 28.

30. A NR comprising a plasmonic metamaterial when used for cell culture and detachment of cells from the cell culture.

31 . A NR according to claim 30 wherein the NR are functionalized to facilitate cell attachment.

32. A NR according to claim 30 or 31 wherein the NR have a high aspect ratio enabling tunability to a desired wavelength for tuning a surface plasmon.

33. A NR according to any one of claims 30 to 32 wherein the aspect ratio enables tuning a surface plasmon resonance in the range of 600nm to 1200nm.

34. A NR according to any one of claims 30 to 33 wherein the NR comprise a plasmonic metamaterial selected from gold, silver, copper, titanium, titanium dioxide, graphene, silicon, and germanium.

35. A NR according to any one of claims 30 to 34 wherein the plasmonic metamaterial is gold. .

36. A NR according to any one of claims 30 to 36 capable of stimulating a surface plasmon by optical stimulation to detach cells from the cell culture.

37. A NR according to claim 36 wherein the NR is capable of stimulating a surface plasmon by exposure to near infra-red (NIR) light.

38. Use of a plasmon for detaching cells from a cell culture substrate according to any one of claims 1 to 8.

39. A cell population comprising cells optionally recovered from a method according to any one of claims 10 to 20.