WO2024161321A1

WO2024161321A1 - Corn endosperm-based biomaterial scaffold

Info

Publication number: WO2024161321A1
Application number: PCT/IB2024/050881
Authority: WO
Inventors: Nicole MAMPHEY; Earl PRINSLOO; Garth ABRAHAMS
Original assignee: Rhodes University
Priority date: 2023-01-31
Filing date: 2024-01-31
Publication date: 2024-08-08

Abstract

The current invention relates to a method for preparing a porous three-dimensional bioscaffold from cooked and expanded corn kernel endosperm as well as to methods for use of the porous three-dimensional bioscaffold from cooked and expanded corn kernel endosperm in the culture of living cells bound on and within the bioscaffold. The method further relates to synthetic tissues comprising the porous three-dimensional bioscaffold and methods of use of such synthetic tissues in medical, diagnostic or synthetic meat applications.

Description

CORN ENDOSPERM-BASED BIOMATERIAL SCAFFOLD

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Tissue engineering involves using biology, engineering, and other disciplines to design and produce a functional replacement for tissues. The tissue engineering process typically involves selecting cells cultured in a specialised bioreactor and seeded in the presence of a suitable scaffold. Scaffolds are materials engineered to resemble the extra-cellular matrix.

Tissue engineering can be used for transplants, drug discovery and drug delivery research and most recently for use in the synthetic meat industry. In vitro tissue engineering models that mimic in vivo conditions can be used screen and remove ineffective drug compounds before reaching the clinical trial stage. Engineered tissues significantly improve drug discovery processes and assist in understanding cell and tissue signalling pathways, gene expression, mechanisms of drug action, drug resistance, and cytotoxicity. These engineered tissues then improve the success rate of clinical trials as more effective drugs are developed. Due to the influence that biomaterials and generated scaffolds have on cells, their behaviour and morphology, it is imperative that the correct biomaterial is selected for tissue engineering (Hench and Polak, 2002; Howard et al., 2008; Khademhosseini et al., 2009; Donahue et al., 2019). The International Organisation for Standardisation (ISO), which regulates the evaluation of biomaterials for tissue engineering applications, has released several standard policies named the ISO-10993 policies. These policies have guidelines and state the requirements for biomaterial and scaffold evaluation. For example, scaffolds need to be tested in their final forms with additional modifications that may be included later to ensure biocompatibility is maintained throughout the tissue engineering process. Additionally, scaffolds are tested under specific parameters and conditions that match physiological conditions.

Three-dimensional (3D) cell culture refers to an artificially made environment in which the cells and their surroundings mimic the 3D nature of innate cells and tissues. These properties indicate cell and extracellular interactions, cellular morphology, and division. Additionally, 3D cell cultures help reduce the use of live animals in the preclinical stages of drug discovery, provide a similar prediction of drug resistance as I ive//7? vivo models, aid in efficient drug-induced hepatotoxicity studies and help reduce the costs of drug discovery on a grander scale.

One example of a 3D cell culturing model is the generation of organised cellular aggregates or spheroids. Spheroids are cells cultured as aggregates and as it mimics tumours, it has been an excellent model for studying tumour growth, formation, and drug targets (Tung et al., 2011 ; Zhuang et al., 2018). Spheroids can be formed through co-culturing with a scaffold (3D biomaterial) or scaffold-free methods such as the hanging drop, suspension, low adhesion plates (used for generation of high numbers of spheroids), static liquid overlay technique, centrifugation, and spinner flasks. However, there are challenges with using spheroids, such as disintegration and deformation of the spheroids, getting drug targets to the core of the spheroid and contact-dependent drugs. Furthermore, not all features in vivo are seen in spheroid models, as there have been reports of hypoxia at the core of the spheroid; the latter effect is dependent on the size of the spheroid. Current 3D spheroid-based drug discovery invasion models are also costly and timeconsuming to create, with uniformity being a significant limitation as there is a lack of control of spheroid size, shape, and cell density (Cavo et al. , 2018; Puls et al. , 2018). There is also questionable relevance of the 3D matrix, their compatibility with high-throughput screening and the mechanical characteristics of the ECM composition.

3D models have been used to investigate cell invasion and cell to cell interactions. Cell invasion refers to the migration and proliferation of cells in response to specific signals such as hormones, growth factors and stimuli through the ECM or to other tissues. This is essential for functions like tissue repair and wound healing. For cell invasion to occur, cell motility and proliferation also need to occur. These two factors subsequently affect the speed of invasion and, therefore, are critical to tissue engineering, where models need to mimic physiological conditions (Ding et al., 2019). Additionally, if errors in cell invasion occur, the consequences are fatal, as seen in cancer metastasis. This leads to a need in understanding cellular invasion through invasion models, as invasion models help develop drug and therapeutic targets for therapy. Numerous models have been designed and developed to study cell invasion due to the utmost importance of this biological function. Unfortunately, many are not physiologically or anatomically accurate due to their design. Some of these include mathematical and bioinformatic models used to interpret cell invasion, which, while helpful, are not physiologically relevant (Veiseh et al., 2011 ; Simpson et al., 2018).

Biomaterials are typically synthetic or natural polymers or a combination of both used in tissue engineering to produce a functional substitute. However, before a biomaterial can be chosen for tissue engineering applications, it must fulfil specific requirements. Biomaterials need to form highly porous scaffolds on which cells can attach, proliferate, differentiate, and form permanent tissues (Howard et al., 2008; Munteanu and Apetrei, 2021 ). They also need to have biomimicry abilities to be biocompatible with cells. Additionally, biomaterials must be biodegradable to suit the proliferating nature of cells and if used in transplantation, must be non-toxic to the immune system of the recipient of the engineered tissue (Griffith and Naughton, 2002; Page et al., 2013; Munteanu and Apetrei, 2021 ). Due to the complexity of the cell environment, the engineering of ideal, suitable biomaterials as a scaffold material for tissue engineering applications remains a core challenge.

Polysaccharides have been investigated as a possible component for biomaterial production for tissue engineering, including alginate, cellulose, and starch (Wahab and Razak, 2016).

Alginate has been used in tissue engineering as scaffolds and hydrogels due to its favourable properties in vivo and in vitro including high biocompatibility, low toxicity, rapid gelation (with the addition of calcium ions), adhesion to cells and good osteogenic differentiation (Castells-Sala et al., 2013; Amini et al., 2021 ). However, there are challenges presented with the use of alginate. These include poor mechanical properties, which result in difficulty controlling the shape of the scaffold and its excessive hydrophilicity, making it soluble in physiological environments. Alginate also has complex degradation capabilities. It is degraded in vivo by enzymolysis, which results in slow, uncontrollable, and incomplete degradation. Additionally, it binds poorly to protein.

As a biomaterial, cellulose is gaining more awareness as it is the most abundant natural polymer found on earth due to its many attractive qualities. It is inexpensive, biocompatible, quickly produced and sourced, has good tensile strength, readily polymerises, and maintains its shape, allowing for control of the shape and structure and elicits a low immune response (Mohamed and Hassabo, 2015; Modulevsky et al., 2016). It has high hydrophilicity, tensile and mechanical strength, and degrades via microbial and fungal activity. This is good for tissue engineering as good hydrophilicity is needed for scaffold cell growth and proliferation. Additionally, cellulose is both macro-and micro-porous, which are attractive qualities for a scaffold for cell seeding as it assists in a biomaterial being able to swell. However, cellulose has low non-specific protein adsorption, especially for mammalian cells (Courtenay et al., 2017). Another disadvantage of using cellulose as a biomaterial would be that degradation is slow as cellulose is degraded by cellulase, an enzyme limited in mammals. Symbiotic bacteria usually produce cellulase. Additionally, the non-enzymatic degradation of cellulose and long term toxicology in vivo has not been extensively studied. Starch is growing in the industry as a potential scaffold for tissue engineering and drug delivery systems (Konstantakos et al., 2019; Tak et al., 2019). Starch is made up of two glucose units: amylopectin and amylose. The amount of amylose present in a starch affects the physicochemical and functional properties, making them more suitable for applications in tissue engineering. Amylose can also be tailored to make its structural applications specific. It is packed into semi-crystalline granules, which can be polygonal, spherical, or lenticular.

Starch scaffolds support attachment, proliferation, and cellular differentiation. Starch fits the requirements needed in biomaterial design; it is biodegradable, biocompatible, and abundant in nature. Unfortunately, starch melts upon solubilisation depending on the origin of the starch, amylose-amylopectin ratio and the size and shape of the granules. This becomes a limitation. Starch as a biomaterial is needed in a fibrous form for particular biomedical applications. This is hard because of its low strength, thermal instability and poor processibility. Methods like electrospinning, which is used to produce fibre using a high-voltage electrical charge, have developed pure gels, scaffolds, hydrogels, and films from starch. However, this is a challenge as starch needs to be modified first with a solvent such as acetic acid or other synthetic polymers. All the solvents and polymers used in these modifications have advantages and disadvantages, so suitable solvents and polymers are still being investigated and optimised for more complex tissue engineering applications (Hemamalini and Dev, 2018; Lin et al., 2021 ).

There is therefore a need for a cost-effective and low-technology tissue engineering solution that has diverse use in fields such as drug discovery and delivery research as well as in the synthetic meat industry.

SUMMARY OF THE INVENTION

According to a first aspect of the invention there is provided a method for preparing a porous three-dimensional bioscaffold from corn kernel endosperm, in particular translucent com kernel endosperm, comprising or consisting of the steps of: a) providing a plurality of com kernels; b) exposing the corn kernels to a dry heat source, including a popcorn maker or microwave source without addition of oil for a period of time suitable to result in a cooked and expanded endosperm and a husk; c) removing the pericarp or husk from the endosperm and discarding it; and d) sterilising the endosperm including by Ultra-Violet (UV) sterilisation, dry heat sterilisation or gamma radiation thereby to form a porous three-dimensional corn kernel endosperm bioscaffold.

Preferably, the com kernels may be Zea mays kernels, in particular Zea mays L var. everta kernels which forms butterfly popcorn endosperm once expanded.

According to a second aspect of the invention there is provided a method for culturing living cells bound to and within a porous three-dimensional com kernel endosperm bioscaffold prepared according to the method of the invention.

The method may comprise the steps of:

(i) providing the porous three-dimensional com kernel endosperm bioscaffold and adding cell culture medium;

(ii) seeding the bioscaffold of step (i) with living cells, including avian, mammalian, fish or invertebrate cells to be cultured;

(iii) incubating the cell culture and bioscaffold, thereby to allow the cells grow and infiltrate the porous bioscaffold such that the living cells are bound to and within the bioscaffold.

According to a further aspect of the invention, there is provided a porous three-dimensional com kernel endosperm bioscaffold prepared according to the method of the invention, for producing an artificial tissue which comprises living cells, including mammalian, avian, fish or invertebrate cells bound to and within the bioscaffold.

According to a further aspect of the invention, there is provided an artificial tissue comprising the porous three-dimensional com kernel endosperm bioscaffold prepared according to the method of the invention. In particular, the artificial mammalian, avian, fish or invertebrate tissue may be used as a synthetic meat product.

According to a further aspect of the invention, there is provided a method for drug or biological medicament discovery, drug or biological medicament resistance assessment and/or drug or biological medicament cytotoxicity assessment with the use of the porous three-dimensional corn kernel endosperm bioscaffold prepared according to the invention which comprises living cells, including mammalian, avian, fish or invertebrate cells bound to and within the bioscaffold.

The method may further include a step of treatment of the living cells with one or more compound(s) to assess the suitability of such compound(s) as a drug or biological medicament for treatment of a subject.

The method may further include a step of infecting the living cells bound to and within the bioscaffold with a pathogen such as a bacterial, viral or fungal pathogen followed by treatment of the infected living cells with one or more compound(s) to assess the suitability of such compounds to inhibit or kill the pathogen without harming the living cells.

According to a further aspect of the invention, there is a provided an ex vivo or in vitro diagnostic method comprising the use of the porous three-dimensional com kernel endosperm bioscaffold prepared according to the invention, or an artificial tissue comprising the porous three-dimensional com kernel endosperm bioscaffold.

According to a further aspect of the invention, there is provided a method of culturing living cells including mammalian, avian, invertebrate or fish cells in a bioreactor with the use of the porous three-dimensional com kernel endosperm bioscaffold prepared according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be further described, by way of examples, with reference to the accompanying drawings. Figure 1 shows XRD patterns showing the intensity in counts of the diffraction angles using 2 0 degrees of potato starch, amylopectin from maize and popcorn endosperm. The x-ray diffraction patterns were recorded using a Bruker D8 Discover, scans analysed using EVA software and the patterns graphed using Excel;

Figure 2A shows the swelling ratio (%) calculated for the popcorn endosperm after incubation with PBS and without PBS for 12 hours at RT. Three independent measurements were conducted for the swelling analyses, and a box and whisker diagram was used to present the data's spread. A: The analysis of the swelling ratio (the percentage of mass increase from the dry weight) of the endosperm over time. B: The analysis of the endosperm's swelling ratio (the percentage of mass increase from the dry weight) with different independent popcorn samples. represents statistical significance detected, where p < 0.05 (p= 3.34 x 10-8). This figure was generated using MS Excel and Statistica software;

Figure 2B shows the swelling ratio (%) calculated for the popcorn endosperm after incubation with media and without media for 12 hours at RT. Three independent measurements were conducted for the swelling analyses, and a box and whisker diagram was used to present the spread of the data. A: The analysis of the swelling ratio (the percentage of mass increase from the dry weight) of the endosperm over time. B: The analysis of the endosperm's swelling ratio (the percentage of mass increase from the dry weight) with different independent popcorn samples. represents statistical significance detected, where p < 0.05 (p = 3.34 x 10-8). This figure was generated using MS Excel and Statistica software;

Figure 3 shows the degradation ratio (%) calculated for the popcorn endosperm after incubation in ddH2O (control) and 0.05 M Tri-Sodium Citrate for 12 hours at RT. Three independent measurements were conducted for the swelling analyses, and a box and whisker diagram was used to present the data's spread. A: The analysis of the endosperm's degradation ratio (the percentage of mass increase from the dry weight) over time. B: The analysis of the endosperm's degradation ratio (the percentage of mass increase from the dry weight) with different independent popcorn samples. represents statistical significance detected, where p < 0.05. This figure was generated using MS Excel and Statistica software;

Figure 4 shows the Cell Index vs Time Curve using obtained from the xCELLigence RTCA software showing the real-time analysis of the infection of RAW 264.7 cells by Mtb for 17 hours. The time curves were averaged for this analysis of three independent replicates, and Excel software was used to represent this data, with error bars representing the standard deviation;

Figure 5 shows a Bar graph of the resazurin fluorescence signal after incubation with HeLa and RAW 264.7 cells seeded on popcorn endosperms. Multiple samples were used. PE stands for Popcorn endosperm samples. A: HeLa seeded cells. INSET Shows assay validation with attached cells. B: RAW 264.7 seeded cells. *Shows statistical significance where p < 0.05, where p= 0.011513 (HeLa); p= 3.34 x 10-8 (RAW 264.7) The graph was generated using MS Excel and Statistica software;

Figure 6 shows Nucleic acid isolation from scaffold;

Figure 7 shows bar graphs illustrating the resazurin fluorescence (Ex 530 nm, Em 590 nm) readings after incubation of com endosperm scaffold with or without C2C12 cells in adherent and/or poly-HEMA coated plates; and

Figure 8 shows SEM micrographs of the ultrastructure of the popcorn endosperm with or without cells and following seeding with C2C12 cells and SEM sample processing for 21 days.

DETAILED DESCRIPTION OF THE INVENTION

The current invention relates to a method for preparing a porous three-dimensional bioscaffold from cooked and expanded corn kernel endosperm as well as to methods for use of the porous three-dimensional bioscaffold from cooked and expanded corn kernel endosperm in the culture of living cells bound on and within the bioscaffold. The method further relates to synthetic tissues comprising the porous three-dimensional bioscaffold and methods of use of such synthetic tissues in medical, diagnostic or synthetic/cultured meat applications. The following description of the invention is provided as an enabling teaching of the invention, is illustrative of the principles of the invention and is not intended to limit the scope of the invention. It will be understood that changes can be made to the embodiment/s depicted and described, while still attaining beneficial results of the present invention. Furthermore, it will be understood that some benefits of the present invention can be attained by selecting some of the features of the present invention without utilising other features. Accordingly, those skilled in the art will recognise that modifications and adaptations to the present invention are possible and can even be desirable in certain circumstances and are a part of the present invention.

The applicant has developed a novel starch-based biomaterial for use in tissue engineering applications. Popcorn (Zea mays L var. everta), a popular snack consumed by many globally, is not only nutritious but made up of a majority of starch, one of the most commonly used polymer biomaterials. Further analysis into the ultrastructure of popcorn showed that its endosperm, the white part of the popcorn, is highly porous and hydrophilic composed of non-allergenic resistant starch. Popcorn is also easy to make, inexpensive and readily available. The applicant therefore went on to study popcorn endosperms' mechanical, structural and biocompatibility abilities. The ultrastructure and surface structure of the popcorn endosperm was characterised through scanning electron microscopy (SEM), micro-computed tomography, and optical microscopy analysis. These showed a highly porous ultrastructure with approximately 36-42 pm large polygonal pockets. The surface structure was rough and characterised by grooves and ridges. The chemical characterisation of the popcorn endosperm was also performed using x-ray diffraction analysis and showed similar diffraction patterns to well-established starch-based samples. The x-ray diffraction, swelling and degradation analysis highlighted the hydrophilic nature of the endosperm, making this an ideal candidate for a biomaterial scaffold used in tissue engineering.

The applicant further conducted biocompatibility investigations with HeLa, RAW 264.7, MDA-MB-231 and C2C12 cells to analyse the depth infiltration of cells into the scaffold, the viability of the infiltrated cells and long-term survival of cells on the scaffold. However it is to be expected that the scaffold may be equally successfully used for the infiltration and growth of other cells known to be grown successfully in vitro, including mammalian, avian, fish and invertebrate cells. Additionally, the scaffold product was assessed for its application in drug discovery through an infection model with RAW 264.7 cells infected with Mycobacterium tuberculosis. It is anticipated that other infection models including different cells and pathogens that are known to be successfully performed using in vitro cell culture could equally be adapted to be performed using the scaffold of the invention. For example, it would equally be expected that the scaffold of the invention could be used for drug or biological medicament discovery, drug or biological medicament resistance assessment and/or drug or biological medicament cytotoxicity assessment using any cells known to be useful for such in vitro studies. Sterility of the popcorn endosperm following microwave expansion was also analysed. Haematoxylin and eosin y-stained sections showed a depth infiltration of at least 126 pm into the scaffold by cells, and resazurin analysis showed viable cells 24 hours after cell-seeding. Extracellular matrix deposits of the cells onto the scaffold were also observed through SEM analysis, and initial testing of the popcorn endosperm as a drug discovery model showed infiltration of RAW 264.7-Mtb infected cells into the scaffold.

The applicant surprisingly demonstrated that popcorn endosperm is a broad range biomaterial for tissue engineering applications, having bioactivity, biocompatibility, and mechanical strength in addition to being inexpensive and easy to use and reproduce.

Popcorn endosperm comes in two forms: opaque endosperm and translucent endosperm. The opaque endosperm forms starch granules that are spherically shaped and separated by air spaces, while the translucent endosperm forms polygonal-shaped starch granules, which are embedded in a protein matrix. The translucent endosperm is more common and was the focus of the applicant’s research. Popcorn consists of protein, lipids (linoleic and oleic acids), fibre, cellulose, water, hemicellulose, and starch; 28% amylose and 72% amylopectin. These percentages may differ slightly due to genetic and growth conditions (Parker et al., 1999; Farahnaky et al., 2013; Freire et al., 2020).

With the rise of novel technologies to assist in tissue engineering and tissue regeneration, the need for biocompatible scaffolds, biomimicry qualities, safe, natural, and inexpensive to use are becoming more valued. The properties of an ideal biomaterial for tissue engineering include biocompatibility, where no toxic degradation products are released, there are antibacterial properties, and the scaffold produces minimal or no inflammatory response. Biodegradability, another important ideal characteristic for the scaffold, is essential where degradation is controlled and can occur biologically or by the metabolic activities of the host. The mechanical properties of the scaffold that are ideal for tissue engineering include the scaffold being able to be modified and still maintain structural integrity. Lastly, the scaffold needs to be bioactive, which results in it being able to interact and adhere to the hosts' tissue, have macropores for cell invasion, micropores and biological factors (such as the free OH groups on cellulose) on the surface area for cellular and protein interaction and have biological factors such as (Turnbull et al., 2018; Qu et al., 2019).

While there have been strides in medicine, microbiology, and biotechnology to meet these needs, particularly with decellularised plant-based scaffolds, there is still a niche and need for novel scaffolds. This is crucial for tissue engineering and regeneration for damaged tissues and global medical diagnostics, drug discovery, and medical research fields as well as in the rapidly developing field of synthetic meat production.

EXAMPLE 1

AIM

To analyse and establish the popcorn endosperms' suitability as an ideal biomaterial for tissue engineering using various characterisation techniques.

OBJECTIVES a. To prepare and process the popcorn endosperm for characterisation through popping and expanding the kernel using a microwave popcorn maker and separating the husks from the endosperm. b. To analyse the infiltration of resazurin and two food colouring dyes into the popcorn endosperm to determine how far into the scaffold substances can infiltrate. c. To analyse the ultrastructure of the popcorn endosperms using optical microscopy visualised with resazurin, phalloidin 594 and auramine/rhodamine dyes. d. To analyse the ultrastructure and porosity of gold-coated popcorn endosperms using SEM. e. To analyse the 3D surface structure of the expanded popcorn endosperm using micro-computed topography micro-CT. f. To characterise the mechanical properties and biocompatibility of the popcorn endosperm through swelling studies in cell culture medium and saline solutions and degradation studies in tri-sodium citrate solution.

METHODS AND MATERIALS

Scaffold Preparation

Popcorn kernels were filled in the popcorn maker silicon scoop. The kernels were popped using a commercial hot air popper as per the popcorn maker instructions until all the kernels had popped out. After that, the husks were removed from the endosperm using forceps. The endosperm was cut into 0.5 or 1 cm³ pieces using forceps and surgical blades that were chemically sterilized in ethanol 70% (v/v) for 5 minutes or fragmented in a coffee grinder until about 0.5 cm³ pieces were produced. The samples were placed in 6-well tissue culture dishes and placed in the Biosafety Cabinet Class I Safety Hood for sterilisation under UV light. For statistical significance, three batches were prepared, and three 1 cm³ individual samples were randomly selected from each batch to fill one well of a 6-well tissue culture plate, one 1 cm³ sample per well in a 24-well culture plate and one 0.5 cm³ sample per well in a 96-well culture plate. The plates were left in the hood until they were dried, then sealed with parafilm and stored at 4°C until use. All camera images were captured with a Xiaomi Note 9 cell phone unless stated otherwise.

Poly-HEMA plate coating

Before use, some culture plates were treated with poly hydroxyl-ethyl-methacrylate (poly- HEMA). Poly-HEMA plates were made by dissolving 1 g of poly-HEMA (Sigma) in 50 ml of 95% (v/v) ethanol on a stirrer for four hours at 65°C. The poly-HEMA solution was then filter-sterilised and used to coat the bottom of 6-well (1 ml) 24-well (500 pl) and 96-well (50 pl) tissue culture plates in the Biosafety Cabinet Class I Safety Hood. The plates were left in the hood until they were dried, then sealed with parafilm and stored at 4°C until use.

Cell Culture and Long-term Cell Growth on the Scaffold Confluent (70%) C2C12 cells (n + P15) cells obtained from lab stocks were cultured in a T75 flask in basal medium, Dulbecco’s Modified Eagle’s Medium (DMEM) containing 4.5 g/L glucose and supplemented with 10% Foetal Bovine Serum (FBS), 1 % Penicillin- Streptomycin-Amphotericin B (PSA) at 37°C with 5% CO2 in a humidified chamber. The cells were sub-cultured when they reached 50-75% confluency using standard trypsinization protocols, centrifuged, enumerated using Cell counter and re-suspended in basal medium. All popcorn endosperms were UV sterilised for 1 hr 30 minutes. Once sterilised, the endosperms were transferred into a 6-well, 24-well or 96-well plate in a Biosafety Class I hood. Basal medium containing 0, 5000, 10000, 20000, 30000, 40000, 50000, 60000, 80000 and 100000 cells was added to 6-well (3 ml) 24-well (1 ml) and 96- well (150 pl) plates followed by incubation for 4, 7 and 21 days at 37°C with 5% CO2 in a humidified chamber under static conditions. Scanning electron microscopy (SEM) analysis, Nucleic acid analysis and Resazurin assays were used to determine scaffold structure and confirm the presence of cells shown by presence of nucleic acid and viable metabolically active cells within the scaffold respectively. Negative controls comprised of wells with media only and popcorn only without any cell seeding. Positive controls comprised of wells with cells only in basal media.

Nucleic acid analysis

Popcorn endosperms were prepared as described above. One millilitre of basal medium containing 0, 5000, 10000, 20000, 50000 and 100000 cells was added to 24-well plates followed by incubation for 7 days at 37°C with 5% CO2 in a humidified chamber under static conditions. The endosperm samples were transferred to clean falcon tubes and washed three times in 20 x PBS volume for 5mins on a rocking platform. Approximately 0.5 g of washed endosperm samples were weighed out and homogenised 10 x in 500 pl PBS in Eppendorf tubes before DNA was isolated from them using plant specific DNeasy ® Plant Mini kit (QIAGEN) and all genomic Quick-DNA TM Miniprep Plus kit as per manufacturer’s instructions. Ten microliters of DNA isolates were mixed with 2 pl of 6 x DNA loading dye, loaded onto a 0.8% agarose gel, before electrophoresing at 80 V for 2 hours, imaged on the ChemiDoc XRS instrument and analysed using Image Lab software (Bio-Rad) manually selecting lanes, bands and range of bands. One clear band from the marker lane was used as a reference for relative quantification. Resazurin Assay

The popcorn endosperm was prepared as stated above. After seeding and incubation at 37°C with 5% CO2 in a humidified chamber for 4, 7 or 21 days, popcorn endosperms were transferred to fresh plates. Samples were washed with PBS before light sensitive resazurin solution was added to samples in fresh media and original plates at a final concentration of 44 pM for incubation at 37°C with 5% CO2 in a humidified chamber for four hours. Controls involved were negative controls of popcorn endosperm with or without basal media i.e growth media only without cells, and positive control of cells only without scaffold. After that, the media/resazurin solution was removed from the wells, put in new plates, and measured the fluorescence at 530 nm excitation and 590 nm emission using the Biotek Synergy Mx plate reader with Gen 5.1.10 software.

Stain Infiltration

The popcorn scaffold was prepared as mentioned above and then incubated at 37°C in a humified incubator with 5% CO2 for one hour with 3 ml of resazurin (1 .5 mg/ml) and 3 ml each of egg yellow and apple green food colouring (MORIS). The food colouring solutions were prepared by placing 200 pl of each into 500 ml (v/v) distilled water. The popcorn endosperm was then sliced open with a scalpel, and images were taken to visualise the depth of infiltration of the stain. Three images were captured per stain. Three independent measurements of each experiment were made, and the images were collated using Inkscape software (Version 1.1 ).

Optical Microscopy

The popcorn scaffold was prepared as mentioned above and then incubated at 37°C in a humified incubator with 5% CO2 for 12 hours in basal medium, Dulbecco’s Modified Eagles Medium (DMEM) + GlutaMAX containing 4.5 g/L glucose supplemented with 10% Foetal Bovine Serum and 1 % Penicillin-Streptomycin and Amphotericin B. The scaffold was then rinsed with PBS, and resazurin, auramine/rhodamine and phalloidin were added according to their specific requirements below. Following the staining, the endosperms were visualised on the EVOS FL Auto 2 using the Brightfield, RF Red (auramine, 460/550 nm/rhodamine, 568/583 and phalloidin, 581/609 nm), filter at 10 X magnification. Three independent measurements of each experiment were made, and the images were collated using Inkscape software (Version (Ver.) 1.1 ). For resazurin staining, 3 ml of 1.5 mg/ml of resazurin was added to the popcorn endosperms and incubated for four hours at 37°C in a humified incubator with 5% CO2, then visualised via brightfield microscopy. Phalloidin staining was done after fixing the endosperms in 4% (v/v) paraformaldehyde (PFA) at RT for 20 min. The PFA was aspirated, and the endosperms were rinsed three times in PBS. Phalloidin Alexa FluorTM 594 stain (1 pl ) was added to the popcorn endosperms for incubation at RT for one hr and 30 min. The stain was aspirated, and the endosperms were washed twice in PBS and then visualised via microscopy. Auramine/Rhodamine staining was performed by fixing the endosperms in PFA for 20 min at RT. The endosperms were then washed with PBS and incubated at 37°C with a 1 :10 dilution of auramine/rhodamine (6 g Auramine 0, 3 g Rhodamine B, 40 g phenol, 300 ml glycerine, 70 ml isopropanol and 140 ml distilled water) stain in PBS for 25 min. The endosperms were decolourised with decolouriser (5 ml hydrochloric acid, 700 ml isopropanol and 300 ml distilled water) for 3 min, rinsed with PBS and then counterstained with potassium permanganate (2.5 g KMNO4 in 500 ml distilled water).

Scanning Electron Microscopy

The popcorn scaffold was prepared as mentioned above and then incubated at 37°C in a humidified incubator with 5% CO2 for 21 days in growth medium, Dulbecco’s Modified Eagles Medium (DMEM) + GlutaMAX containing 4.5 g/L glucose supplemented with 10% Foetal Bovine Serum and 1 % Penicillin-Streptomycin and Amphotericin B. The endosperms were then prepared for SEM analysis by washing with 0.1 M phosphate buffer for 5 minutes and fixing in glutaraldehyde overnight. This was followed by dehydration consecutively in increasing ethanol concentrations (30%, 50%, 70%, 80%, 90% and 100% v/v) for five minutes each. The dehydrated samples were then dried in a critical point dryer (CPD) for approximately two hours while immersed in 100% ethanol. All SEM samples were gold-coated with a Quorum Q150R S gold sputter for approximately 10 minutes, and the ultrastructure was visualised using the TESCAN Vega TS 5136LM scanning electron microscope. Micrographs were recorded at an acceleration voltage of 20 kV, analysed using Vega TC software. Negative controls comprised of fresh dry endosperm and endosperm only without any cell seeding incubated in basal/growth medium for 21 days.

Micro-Computed Tomography Analysis The popcorn endosperm was prepared as stated above. After that, the popcorn endosperms were incubated at 37°C in a humified incubator with 5% CO2 for 12 hours in a basal medium and immersed in PBS until scanning began. The endosperm was then scanned using Bruker SkyScan 1173 high-energy Micro-CT scanning system for an hour and 30 minutes, generating multiple cross-section images at 16 pm steps. Sample images representing the characteristics on the surface of the popcorn endosperm were selected and analysed using CTvox (Ver. 3.3.1 ) software. The macropores were then analysed using segmentation analysis on Imaged software (Package 1 ,53k).

X-Ray Diffraction Analysis

The popcorn endosperm was prepared as mentioned above. To ensure the endosperms were in a powdered form as used in x-ray diffraction analysis, the endosperms were emersed in liquid nitrogen and ground using a glass mortar and pestle until completely powdered. Cellulose, potato starch and amylopectin from maize were scanned with the ground popcorn endosperm as a reference, and all samples were placed on a silicon wafer slide. The x-ray diffraction patterns were recorded on a Bruker D8 Discover equipped with a proportional counter, using Cu-Ka radiation (A = 1.5405 A), nickel filter. The data from the scans were analysed using EVA (evaluation curve fitting) software (Ver 6) and collected in the range from 2 9 = 10° to 100°, scanning at 1.5° min-1 with a filter time-constant of 0.38 s per step and a slit width of 6.0 mm. Each diffraction pattern was baseline corrected by subtracting a spline function fitted to the curved background. Microsoft (MS) Excel was used to visualise and present the x-ray diffraction patterns.

Swelling Analysis

The swelling ability of the popcorn endosperm was carried out in a 24-well plate following preparation, as mentioned above. The popcorn endosperms were placed in a well individually with 1 ml of PBS (1.37 x 105 mM NaCI, 2.7 x 103 mM KCI, 1 x 104 mM Na2HPO4, 1 .8 x 103 mM KH2PO4) and basal medium. No PBS was added to the negative control wells. The plate was incubated at room temperature (RT), and the initial mass (grams) was recorded, followed by the initial immersion in basal medium or PBS (time (t)= 0), after 6 hours (t= 6) and after 12 hours (t= 12) of incubation. Three independent measurements of each experiment were made. The average mass of the endosperms was used to calculate the swelling ratio over 12 hours:

Swelling Ratio=(Mass at T₁₂ -Mass at T₀)Mass at T₀ x 100... 1, where “Mass at T12” is the mass recorded after 12 hours of incubation and “Mass at To” is the dry weight.

Following this, a time-lapse analysis was performed to observe the swelling of the endosperms over 72 hours in a 24-well plate following preparation, as mentioned above. The popcorn endosperms were placed in a well individually with no fluid (control) and the addition of 2 ml of PBS, distilled water and basal medium, incubated at 37°C in a humified incubator with 5% CO2. Images were taken at intervals of one hour, three hours, 24 hours, 48 hours, and 72 hours. Three independent measurements of each experiment were made, and the images were collated using Inkscape software (Version 1.1 ).

Degradation Analysis

The degradation ability of the popcorn endosperm was carried out after the preparation of the endosperm, as mentioned above. The endosperms were then placed in individual wells in a 24-well plate with 1 ml of 0.05 M Tri-Sodium Citrate, with PBS incubation serving as the negative control. The plate was incubated at room temperature (RT), and the initial mass (grams) was recorded, followed by the initial immersion (t= 0) in 0.05 M tri-sodium citrate/PBS, after 6 hours and after 12 hours of incubation. Three independent measurements of each experiment were made. The mass of the endosperms was averaged, and the mass loss/gained by the endosperms was plotted over 12 hours.

Data Analysis

The data obtained from the three independent experiments were analysed to determine the statistical differences between the various samples and times. The one-way ANOVA (analysis of variance) test was used for the swelling analysis, and a paired t-test for the degradation analysis and a p-value of < 0.05 were considered significant. Microsoft Excel software and TIBCO Statistica (14.0) were used to generate graphs and calculate statistical significance. RESULTS AND DISCUSSION

Scaffold Preparation

The preparation of the popcorn scaffold was carried out using commercially sourced popcorn kernels and a popcorn maker to induce the expansion of the popcorn endosperm from the kernel based solely on the endosperms' endogenous water. The process of scaffold preparation was documented, and images were captured using a Xiaomi Note 9 phone camera. A ruler was used for size approximation to display how the size of the kernel changes as expansion occurs. The kernel shape after expansion has a butterfly morphology and expanded almost three times the kernel size as shown by the ruler. The pericarp was still intact with the endosperm and was then removed and discarded. The endosperm’s size was the same as the kernel size before expansion, highlighting how much mass the pericarp adds to the endosperm. The endosperms were then placed in tissue culture plates, ready for various characterisation methods and determination of their potential as an ideal biomaterial for tissue engineering applications.

Kernels that produce the butterfly-shaped popcorn endosperm were chosen because these have fewer husks, are more tender and have a larger surface area in comparison to the mushroom-shaped popcorn endosperm kernel. These characteristics are essential when considering a biomaterial for tissue engineering applications because fewer husks would result in less scaffold manipulation and a quicker preparation time. The tenderness of the endosperm could prove suitable for biocompatibility and biodegradability, especially in the application for hard tissue engineering, which may not require as many modifications to a scaffold. Lastly, the larger surface area is a vital feature for tissue engineering.

Further experiments were performed using microwave expansion of the kernels as it is more efficient and cost-effective. Additionally, oil has been shown to add extra fatty acids when made with popcorn, and this could potentially disrupt scaffold integrity as this decreases the expansion volume of the kernel as well as affect its hydrophilicity. This highlights an important, ideal scaffold characteristic that popcorn endosperm has of being cost-effective, easy to make and reproduce. As the experiment relied solely on the endogenous water content in the popcorn endosperm itself for expansion, additional water was not needed. This shows that the endosperm's hydrophilic characteristics in further experiments are not due to external modifications. With many scaffolds needing additional manipulation to increase their hydrophilic characteristics, this also shows that the popcorn endosperm possibly has another ideal characteristic for biomaterials used in tissue engineering applications. Following expansion, husks of the expanded popcorn were removed from the endosperm with forceps. The kernel was shown to expand almost three times its original size. Processed kernels that are similar in size were chosen for characterisation to assist with determining statistical significance during characterisation and validation.

Following the process of the scaffold preparation, which entailed the expansion of the kernels and husk removal, the popcorn endosperm was ready to be characterised and validated for its application as a biomaterial and scaffold for tissue engineering applications.

Stain Infiltration Analysis

The popcorn scaffold was prepared using the procedure above and then incubated at 37°C for one hour with resazurin and two types of food colouring, egg yellow and apple green. The popcorn endosperm was then dissected with a scalpel, and images were taken to visualise the inside of the endosperm to investigate the depth of infiltration of the stain. Two popcorn endosperms were sliced down the middle into two halves to observe the depth of infiltration of the food colouring by the endosperm, showing that the stain infiltrated the entire endosperm. The same procedure was carried out with egg yellow, another food colourant, as a differential and resazurin, a commonly used dye for cell viability studies. The same depth infiltration with the green food colourant was observed with the resazurin dye and with the egg yellow food colourant (results not shown).

This was a crucial step to ensure that the characterisation techniques, such as cytotoxicity studies, would be of the surface of the endosperm, which is essential and of the inner 3D structure and matrices of the endosperm. By showing that the various dyes can infiltrate the popcorn endosperm, a case for biocompatibility can be built as this shows that nutrients and oxygen can potentially diffuse in and out of the endosperm, circumventing anoxia which is often observed in various 3D models. This also shows a potential for cell migration and proliferation into the scaffold, since the dyes were able to infiltrate the entire endosperm, the same can occur with cells.

Optical Microscopy Analysis

The preparation of the popcorn scaffold was performed as previously reported. The popcorn endosperms were incubated at 37°C in a humified incubator with 5% CO2 for 12 hours in basal medium. Various preparation methods were used, and staining with resazurin, auramine-rhodamine and phalloidin 594 was performed to visualise the field view microstructure of the popcorn endosperm using the EVOS FL Auto 2 microscope at 10x magnification. Surface scan images of the stained, unsliced popcorn endosperm were taken, which shows the whole endosperm's field view intact. The auramine-rhodamine staining of the popcorn endosperm shows polygonal pockets can be observed on the surface of the endosperm (results not shown). As observed with the auramine-rhodamine stain, the polygonal pockets can also be seen with the phalloidin stain.

Scanning Electron Microscopy Analysis

The ultrastructure of the popcorn endosperm was investigated after the microwave expansion of the popcorn endosperm and incubation in cell media for 12 hours. The samples were fixed with glutaraldehyde overnight, dehydrated with increasing ethanol concentrations, dried with a critical point drier, coated with gold, and then analysed using the TESCAN Vega TS 5136LM scanning electron microscope. Micrographs were recorded at an acceleration voltage of 20 kV and analysed using Vega TC and Inkscape software. The results show a 3D image of the polygonal surface structure observed above. Popcorn endosperm was found to have porous microstructures and pockets, an ideal characteristic for a biomaterial used in tissue applications. The ultrastructure of the endosperm is composed of polygonal structures that are packed together, forming a natural scaffold and highlights the possibility of the endosperms’ usage as a biomaterial based on the observed pore size. The size of the endosperm pockets was calculated to be 42.875 pm in diameter using the scale bar of the SEM imaging software and Image J segmentation analysis. This could potentially be another plant-based biomaterial that can aid in improving the mechanical properties of alginate. Additionally, this is suitable for cell types such as fibroblasts and nerve cells, which prefer small pores for adhesion and proliferation. However, for its use for tissue engineering applications, the endosperm would need to be biocompatible, and one of the first ways of determining this would be incubation in cell media for some time. This was done for 12 hours, and the ultrastructure was visualised to observe any adverse effects. Unfortunately, the SEM cannot be used on wet samples, so the popcorn endosperm was further processed through a fixation with glutaraldehyde overnight and then dehydration with increasing ethanol concentrations for 5 minutes each (50%, 70%, 80%, 90% and 100%). Following the dehydration process, the popcorn endosperms were dried using a critical point dryer and then coated with gold to ensure that the microscope used in the SEM process observed a signal that could be interpreted and analysed. The porous structure of the popcorn endosperm can still be observed following the processing step. Here, the size of the endosperm pockets was calculated to be 34.7748 pm in diameter. Compared with the unprocessed endosperms, with a diameter of 42.875 pm, this shows the strength of the popcorn endosperm to withstand severe processing. Even though there is a reduction in the size of the pockets, it is not by a considerable amount (8.1002 pm difference). This highlights a good characteristic of this biomaterial to possess. Furthermore, the ability of the porous structure to withstand these disruptions highlights the materials' structural integrity, another good characteristic that a biomaterial should possess.

Micro-Ct Analysis

The surface structure of the popcorn endosperm was investigated after the microwave expansion of the popcorn endosperm and incubation in basal media for 12 hours. The samples were then immersed in PBS for an additional 24 hours for transporting to the micro CT facilities and then analysed using the Bruker SkyScan 1173 micro-computed tomography scanning system. Three-dimensional scans were analysed and constructed using CTVox software and collated together using Inkscape software. A 3D constructed image of the popcorn endosperm was generated with the frontal view of the structure showing the shape and macro-view morphology of the popcorn endosperm. This view shows grooves and ridges present on the endosperm. The bottom view of the endosperm shows the grooves and ridges present on the frontal view and highlights visible macropores on and in the structure of the popcorn endosperm (data not shown). Various angles of the sliced endosperm were generated showing grooves, ridges and macropores. This further shows that the macropores observed on the bottom view of the popcorn endosperm are not just external but are found within the internal structure of the endosperm as well.

To assist with quantifying the size of these pores, a 2D image of the slices was reconstructed of the endosperm. The surface structure of the popcorn endosperm, as observed through the micro-CT scans, shows a surface roughness in the form of grooves and ridges, which could assist with biocompatibility as it would provide cells and proteins with a surface to adhere to and enhance the adhesion abilities of the scaffold.

Pores observed on the bottom surface of the endosperm seem to be consistent across the entire structure, as these are visible in all slices in multiple planes of the structure. It is also observable that these pores are in different sizes and shapes yet are also different to the pores observed on the ultrastructure that is uniform, tightly packed (when not disrupted) and polygonal. The large pores go through the entire endosperm.

Additionally, the slices illustrated macropores and micropores found in the endosperm structure and within. These pores would be an excellent quality to have for nutrient and oxygen exchange and diffusion. The pores were quantified, showing various sizes found within the popcorn endosperm. The smallest pore was 39.000 pm wide and the largest pore was 170.763 pm. This pore was not vast in diameter but more extended, length-wise. The variety in these pore sizes could prove advantageous for tissue engineering applications for both soft and hard tissues. For example, for cells like fibroblasts which prefer small pores for infiltration, this could be useful for soft tissue applications and for cells like osteoblasts which prefer large pores for infiltration, this could also be a scaffold used in osteoblast tissue engineering applications. Additionally, the variety of these pore sizes could be beneficial for co-culture cell models. The information displayed by the micro- CT images also confirms the potential of the scaffold to be used as a biomaterial due to the porous nature of the material and the surface roughness, important characteristic, and feature of a biocompatible biomaterial for tissue engineering application.

X-Ray Diffraction

The crystallinity of the popcorn endosperm was analysed using an x-ray diffractor. The popcorn endosperm was processed after microwave expansion and immersed in liquid nitrogen, and ground into powder using a mortar and pestle. This was because x-ray diffraction samples were analysed in powdered form, so the popcorn endosperm was frozen and ground for x-ray diffraction analysis. As a reference, cellulose, potato starch and amylopectin from maize were also analysed using the x-ray diffractor as they are well- established and characterised in literature. The samples' x-ray diffraction patterns were recorded on a Broker D8 Discover. The scans were analysed using EVA and graphed using Excel. The intensity (counts) of the diffraction angle (20) of the crystalline structures in the individual samples are plotted to highlight the similarities and differences between the crystalline structure of amylopectin from maize and potato starch and the popcorn endosperm. As can be seen in Figure 1 , the three samples show peaks around 20°. However, the peaks of amylopectin from maize and potato starch are sharper and more intense than those of the popcorn endosperm. Popcorn endosperm and potato starch have one intense peak, whereas amylopectin from maize has two intense peaks. This points to the similarity of the crystalline structure of popcorn endosperm more to the potato starch than amylopectin from maize.

X-ray diffraction patterns assist in determining the structure, crystallinity, and hydrophilicity of samples. In tissue engineering applications, it is used to characterise the structure of biomaterials to determine and establish suitability for various applications. As amylopectin and potato starch are well-established and characterised substances, their diffraction patterns were analysed and used as reference material for the popcorn endosperm to identify its diffraction patterns. Amylopectin from maize showed two significant peaks at 20° and 25° of the intensity of approximately 5 500 and 4 800 counts, respectively. Potato starch had a single prominent peak at 20° of an intensity count of approximately 5 500. These peaks are in accordance with literature, which shows starch samples to have similar diffraction angles and peak intensity with slight differences depending on the amylopectin/amylose ratio and the origin of the starch. Potato starch, a B-type of starch, has shown peaks at 20-22°.

Starches classed as B-types have high amylose concentration and are more hydrophilic as theory structures are loosely packed and open to interacting with more water molecules. Amylopectin is highly branched, and its structure is more compact, in accordance with A- types of starches, which have high amylopectin content. A-types have been shown to have peaks at 15° and 23° and are more compact and less soluble than B-types due to the compact structure not being able to interact with more water molecules. There is also a C- type classification of starches, which is an intermediary between the A-type and B-type, with peaks around 15°, 17°, 19° and 22° and an intermediary water solubility ability (Martens et al., 2018; Govindaraju et al., 2021 ).

The popcorn endosperm sample had a less intense peak than the two starch samples at approximately 1 600 counts but had a similar diffraction angle of 20°. The popcorn endosperm appears to be a C-type starch.

X-ray diffraction patterns with high peak intensities and well-resolved peaks point to powder crystallisation. This was observed with the amylopectin and potato starch samples, with almost 5 x higher peak intensity and more resolved peaks than popcorn endosperm. This can be alluded to the commercial production of these substances by various manufacturers that produce highly refined and crystalised amylopectin and potato starch for research and diagnostic use compared to the popcorn endosperm sample, which had not been refined and processed as would occur on a large scale in a company. Figure 1 also shows potato starch and amylopectin with peaks of approximately 1 000 counts at 31 °, whereas the popcorn endosperm has no peaks at this angle.

Swelling Analysis

The swelling abilities of the expanded popcorn endosperms were tested by incubating the endosperms in PBS, basal media, and no PBS/media (control) at RT for 12 hours. The mass in grams of the endosperm was recorded at various stages; the dry weight, after the initial immersion in media/PBS, after 6 hours, and after 12 hours of incubation. The swelling ratio of each endosperm was calculated using equation 1 , compared and analysed. Timelapse images of the swelling analysis for 72 hours at various time points of incubation: one hour, four hours, 24 hours, 48 hours, and 72 hours show the changes to the popcorn endosperms' structure and shape over time and were used as a qualitative analysis of swelling.

Figures 2A and 2B show the summary box plots used to represent the results of the swelling analysis at t= 6. The box plot of A is for the control, which was not immersed in any medium. The box plots of B, C and D, are independent popcorn samples immersed in medium for swelling analysis. Figure 2A was conducted in PBS, and Figure 2B in basal medium. These figures were used to quantitatively analyse the changes that occur to the popcorn endosperms mass during swelling.

A biomaterial's swelling and degradation abilities are essential factors to consider for its suitability for tissue engineering applications as these factors affect pore size, mechanical properties, and diffusion abilities. Previous research on popcorn endosperm's water content and solubility has mainly been on the milled kernels, not the endosperm, as the kernels are often used as flour. It was observed that the control popcorn endosperms with no media were their original standard size as from when it was prepared. However, after an hour of incubation, the popcorn endosperms had shrunk noticeably. This could be due to the temperature, as while the other endosperms were hydrated by their mediums, the control was not. This remained the same over the rest of the time and did not shrink/expand. For the popcorn endosperms immersed in a liquid medium, the initial swelling increase can be observed across all three mediums of PBS, distilled water and basal medium. This shows the swelling of the popcorn increases, resulting in a mass change as the endosperm is filled and absorbs liquid. The popcorn endosperm is hydrophilic and does not fall apart or disintegrate over the time observed. This shows structural integrity, which can be critical for soft tissue engineering applications with soft tissues with cardiac and nerve tissue and hard tissue engineering like bone and cartilage, which require a structurally sound and rigid scaffold to form niches (Garcia et al., 2021 ).

The swelling analysis was performed over 12 hours as a preliminary test. An ANOVA statistical test was run in conjunction with a box-and-whisker analysis to determine if there was a difference between the swelling ratio at t= 6 and t=12. The result of the ANOVA was p > 0.05 (p= 0.745328), showing that there was no statistical difference between the swelling ratio at the sixth hour and twelfth hour mark. As such, t= 6 was used for all statistical analysis moving forward as it had the most significant mean (t=6: 880.828; t=12: 850). This could be inferred from the swelling ratio reaching the maximum at t= 6 and then plateauing out. In Figure 2A and Figure 2B, the percentage of the mass of the popcorn endosperm that increased at the sixth hour of incubation as the swelling ratio is shown. This mass change is due to an increase in the swelling ability of the endosperm. PBS was used for the initial test due to its pH neutrality (Figure 2A). This was followed by testing in the basal medium as it is the basal media used in cell culture applications and represents physiological conditions (Figure 2B). Due to the sample population (n = 18), the box and whisker plot was chosen to show the summary and spread of the data as it can represent large amounts of data quickly and more concisely.

It is observed in Figures 2A and 2B that the initial immersion in liquid increases the mass significantly as it swells up, but after that, it plateaus. The popcorn endosperms swell up from 0% (initial mass, box A) to almost 10 x the initial mass when immersed in liquid (boxes B to D). It is noted that there are no significant differences between samples B to D, which were the sample tests. There is a statistical significance between samples A and samples B to D. This is firstly shown on the plot by the box and whisker plots not overlapping. The ANOVA results showed a statistical significance with p= 3.34 x 10-8 and p= 3.34 x 10-8 (Figure 2B), which is less than 0.05, the statistical significance threshold. Due to this, a post-test had to be performed to determine which groups were statistically different. This showed that sample A was statistically different to samples B to D, but samples B to D were not different. This follows the trend observed in Figure 2A and Figure 2B, where there was no difference in the samples over time after the initial immersion in liquid.

To determine if there is a statistical difference between the PBS and basal media used, an ANOVA test was conducted between t-=6 of both the PBS and DMEM swelling ratios; while the means of the PBS was slightly higher than that of the media, there was no statistical difference observed between the two fluids. This highlights the biocompatible nature of the popcorn endosperm as it is suitable in both basal medium and PBS as it is hydrophilic and can enhance protein and cell attachment.

Degradation Analysis

The degradation abilities of the expanded popcorn endosperms were tested by incubating the endosperms in ddH2O (control) and 0.05 M Tri-Sodium Citrate at RT for 12 hours. The mass in grams of the endosperms was recorded at various stages; the dry weight, after the initial immersion in PBS/tri-sodium citrate (time = 0), after 6 hours and after 12 hours of incubation. The degradation ratio of each endosperm was calculated, compared, and analysed in Figure 3 below. In Figure 3 A, the summary box plots used to represent the results of the degradation analysis at t=6 are shown. The box plot of A is of the control, and this presents popcorn endosperms that were immersed in PBS. The box plots of B, C, and D are independent popcorn endosperm samples immersed in tri-sodium citrate. As degradation refers to a loss of mass of a sample over time, box plots summaries of the increase/decrease in mass over time, being twelve hours, is also shown in Figure 3 B. This reveals whether there was a significant mass loss over time and if the sample was degrading.

Degradation of biomaterials is essential to allow cells to enter the matrix during proliferation and to ensure the cells can remodel and form functional, nascent tissues (Diba et al., 2021 ). The degradation analysis shows an increase in weight size after immersion in the 0.05 M Tri-Sodium Citrate. No significant difference was observed between the degradation profiles and changes in weights over time and with the independent samples (p > 0.05). Trisodium citrate was used to test for degradation capabilities and potential. Due to the sample population (n = 18), the box and whisker plot was chosen to show the summary and spread of the data as done with the swelling ratio. In Figures 3 A and B, the initial immersion in liquid increases the mass significantly as it swells up, but after that, it plateaus out. The popcorn endosperms swell up from 0% (dry weight) to almost 10 x the dry weight when immersed in liquid. This is also visually confirmed by the box and whisker plot overlapping, which indicates no statistical significance between the two.

The ANOVA statistical test was used after that and confirmed no statistical difference between the various time points over the incubation period. Because the dry weight of the popcorn compared to the wet weight would show a statistical difference, this was not plotted. Additionally, PBS was used for sample A as the control, and sodium tri-citrate was used for the test samples B to D. There was no statistical significance between these two, highlighting no difference between the two mediums used to test for degradation of the endosperm.

DISCUSSION

Various methods have been used in characterising the popcorn endosperm as a scaffold biomaterial for tissue engineering applications. The preparation and processing of the endosperm show that the method is easy, quick, and efficient to perform. This can also be done on a large scale, which is feasible. This is promising for a potential biomaterial being evaluated as cost-effectiveness, ease of use and manufacturing, and reproducibility are ideal characteristics of a biomaterial. Table 1 below shows the major components involved in the preparation of the popcorn endosperm and the respective prices. In the timeline of this study (2/ years), only one microwave popcorn maker (R75) and 1 bags of popcorn kernels (R20 each) were used. Barring the variable costs such as microwave voltage and electricity use, which would depend on where the popcorn is made, and the microwave, the total price of the major components involved was R115. This shows the costeffectiveness and sustainability (as only one popcorn maker was used) of the popcorn endosperm as an ideal biomaterial scaffold to be used in tissue engineering applications.

Table 1: Cost-analysis of the major components involved in preparing popcorn endosperms used in this study.

After preparing the popcorn endosperms, infiltration tests were performed to observe how deep substances would infiltrate the scaffold and if it would be permeable for stains and eventually cells during cytotoxicity testing. FDA approved food colourants and resazurin, a commonly used cell viability stain, were used to stain the endosperms to investigate their infiltration capabilities. As both the food colourants and resazurin stained the entire endosperms, which was shown when the endosperms were dissected to observe the inner core, another ideal characteristic of a biomaterial was met- the ability of a biomaterial to facilitate the transport of nutrients of oxygen through a scaffold made up of the biomaterial. Optical microscopy was used next to obtain a field view and surface scan of the structure of the popcorn endosperm. The images showed polygonal structures on the surface of the popcorn endosperm and pointed to a porous ultrastructure. While these stains used in the optical microscopy are non-specific, they bind to long-chain carbohydrates. These polygonal structures were validated using a scanning electron microscope. The ultrastructure of the popcorn endosperm was characterised and evaluated. The ultrastructure was shown to have polygonal structures that could form a scaffold for cells to attach to and start to form niches. These structures are porous, and the pores are about 50 pm in diameter large and could aid in this biocompatibility nature, an ideal characteristic of a biomaterial. Even though further processing of the scaffold (swelling, fixation, dehydration, and critical drying) caused some damage to the ultrastructure, the polygonal structures can still be observed and used for cell adhesion quantitively; there was no significant difference between the pore size pre (42.875pm) and post (34.7748 pm) immersion in solution. This points to the structural rigidity and integrity of the popcorn endosperm, an attractive quality of a biomaterial. While the SEM micrographs showed the ultrastructure of the endosperm, the micro-CT scans highlighted the surface topography of the endosperm as well as on multiple planes in its internal structure.

The micro-CT scans showed that the surface of the popcorn had grooves and ridges. Additionally, there were pores and holes observed on the surface. These features are promising as they can further enhance the cell adhesion quality of the popcorn endosperm and assist in protein to cell interactions. The micro-CT images also highlighted the porous nature of the endosperm, which was a different type of porosity as observed by the SEM. The macro-and micro-pores observed on the micro-CT images could further aid in the biocompatibility nature of the popcorn endosperm as the pores could provide a channel for oxygen and nutrient exchange and uptake that cells require during metabolic processes. This is an important feature and should be investigated further through morphological and structural testing. As a popular snack consumed by many, the popcorn endosperm shows promise as it is already naturally degraded and consumed by the body with no adverse effects.

X-ray diffraction patterns of amylopectin from maize, potato starch, cellulose and popcorn endosperm were analysed to determine the crystallinity of the structure and how packed it is and the samples’ water solubility. The popcorn endosperm peaks also show similarities to amylopectin and potato starch. This shows that the popcorn endosperm has amylose and amylopectin units in its structure and can therefore be classified as a starch-based biomaterial. Additionally, the similarities between the peaks are in accordance with literature and highlight the solubility and hydrophilicity of starch samples, which can be inferred to the popcorn endosperm. However, at the shared diffraction angle of 20°, the endosperm sample had weak peak intensities (1 600) than the potato starch and the amylopectin (both at 5 500), which could be due to the impurity of the sample. Compared to the three starch-based samples, cellulose also peaked at 20° but had a much higher intensity at approximately 20 000. This could be due to cellulose's highly crystalline structure due to its high number of OH bonds.

The ability of the popcorn endosperm to swell and degrade was tested. The swelling abilities are observed both visually and statistically, as when immersed in liquid, the endosperm swells up in the well and is significantly heavier, indicating its ability to take up water. Additionally, the structure of the popcorn endosperm was maintained over a significant time. The popcorn endosperm maintained its structural rigidity and integrity after 72 hours in medium. This is another ideal and attractive characteristic of biomaterials that the popcorn endosperm has.

Biomaterials are selected based on their porous nature, biocompatibility, and biodegradability, allowing for cell encapsulation (Turnbull et al., 2018). Therefore, following the characterisation of its mechanical, structural, and topographical properties, the validation of the popcorn endosperm with a well-established cell model was the next step in the process. A summary of this is shown in Table 2 below,

Table 2: Summary of the ideal characteristics of a scaffold tested so far on the popcorn endosperm

EXAMPLE 2 The Validation of the Popcorn Endosperm as a Biocompatible Biomaterial with HeLa, RAW 264.7, MDA-MB-231 and C2C12 cell models

INTRODUCTION

The biocompatibility of a scaffold should result in an appropriate response when used in vivo. It should not cause injury or damage to the host or be cytotoxic. As such, cell models are used to validate scaffolds and biomaterials for their biocompatibility. Biocompatibility does not always result in cell attachment and adherence. This results in cells having issues attaching and proliferating on a scaffold as they would do to a tissue culture plate/dish or in vivo. A scaffold for tissue engineering applications should enable cell growth and extracellular matrix formation (Chan and Leong, 2008; Thu-Hien et al., 2018; Pina et al., 2019). In testing for biocompatibility, direct methods such as directly seeding cells onto scaffolds and indirect methods of incubating cell culture in media extracts of the scaffold can be utilised. For evaluation of cytotoxicity, which shows whether material is biocompatible or not, methods such as SEM imaging, haematoxylin/eosin histology staining and various cell viability assays such as MTT, MTS and resazurin are standard and frequently utilised (Cannella et al., 2020; Bar-Shai et al., 2021 ).

The use of cell models such as immortal cell lines has been a welcomed advancement in biology. They are essential because of the insights and observations in various biological applications and are critical in the preliminary stages of characterisation and validation of biomaterials and scaffolds. Additionally, they are used to study biological properties and have desirable qualities. From being cost-effective, easy to use and reproduce, and unlimited availability, there are also pure, well-characterised populations of these cell lines. This attribute makes it easier for post-seeding analysis as the populations are homogenous. They also pass the ethical concerns involved with using animal and human tissue. These cell models are invaluable tools for academic and industrial applications and have been used in drug development, cancer research and tissue engineering applications. Additionally, they are helpful as primary cell lines have low proliferation rates and must be used in the early stages of culturing due to their tendency to lose their structural, functional and self-renewal properties the longer they are passaged. Examples of these cell models include epithelial cell lines such as HeLa cells. These cell lines have good adhesion properties, are used in many medical biocompatibility studies, and have a high growth rate.

HeLa cells are the oldest and most widely distributed permanent human cell line. HeLa cells are widely available and used in various applications due to their ease of use and culture as they propagate exceptionally well, especially in tissue culturing conditions. In addition to HeLa cells more cell lines used for studying various diseases and for biocompatibility and cytotoxicity assays have been developed, depending on the potential function of that biomaterial. Examples of these include the HepG2 cell line, which is a human liver cell line, used to investigate the drug metabolism and hepatoxicity of the liver, as well as biocompatibility assays for potential hepatocyte scaffolds (Nikolic et al., 2018; Brooks et al., 2021 ). RAW 264.7 cells have also been used to test and investigate cellular inflammatory responses to numerous applications, including biomaterial characterisation (Lucy et al., 2022). As most preliminary scaffold characterisations and biocompatibility validation in tissue engineering start with HeLa cells, the initial biocompatibility testing of the popcorn endosperm in this study also utilised HeLa cells.

Murine RAW 264.7 is also an established cell line composed of monocyte cells, which can be induced into macrophage-like cells and used as an appropriate cell model for macrophages for more than 40 years in research and academia. Due to the ability of RAW 264.7 cells to provide a macrophage-like model for biological applications, its phenotype and functional stability have been investigated to ensure it is functional and viable to use for research. To induce RAW 264.7 monocyte cells to macrophages, lipopolysaccharide (LPS) is used, or a microbe such as Mtb can infect the RAW 264.7 cells resulting in an immune response and subsequent morphological change. This aids in studying the antiinflammatory properties of drugs, monitoring cellular responses to microbes and their effects on cells and general cellular responses. In addition to these many uses, RAW 264.7 cells are not complicated to culture and propagate. The culturing and passaging techniques are also easy, and the cells are homogenous and widely available. This makes the cell line a suitable candidate for studying the inflammatory response and cell viability of the popcorn endosperm. C2C12 are mouse skeletal muscle cells (myoblasts) that can be coaxed to differentiate into mature muscle fibres, allowing the cell line to act as a model for understanding muscle biology as well as to investigate the effects of muscle cell attachment, fusion and formation of muscle fibres in cellular agriculture applications.

The metastatic breast cancer model, MDA-MB-231 , isolated from a pleural effusion, is used as a model for triple negative breast cancer (lacking key estrogen progesterone and human epidermal growth factor 2 receptors). The cells thus represent a model for studying recalcitrant breast cancers that resist typical chemotherapeutic interventions. As a metastatic, highly aggressive model, the cells are an ideal system to study invasion and migration on and in 3D scaffolds such as the endosperm model described here.

AIM

To validate the biocompatibility of the popcorn endosperm for tissue engineering and regeneration applications using well-established cell models of HeLa and RAW 264.7 cells.

OBJECTIVES a. To analyse the cell adherence and invasion into the ultrastructure of the popcorn endosperm with HeLa and RAW 264.7 cells using scanning electron microscopy (SEM). b. To observe the adherence abilities of the surface structure of the expanded popcorn endosperm when seeded with unfixed HeLa and RAW 264.7 cells using microcomputed tomography (Micro-CT). c. To observe and analyse the infiltration and invasion of HeLa and RAW 264.7 cells stained with haematoxylin and eosin y in the pores of the popcorn endosperm using histology analysis after microtome sectioning. d. To analyse the cellular attachment and viability of HeLa and RAW 264.7 cells on the popcorn endosperm as a scaffold through resazurin absorbance quantification analysis. e. To investigate long-term cell culture of MDA-MB-231 and C2C12 cells on the popcorn endosperm scaffold through live-dead viability staining in non-adherent conditions. f. To validate the popcorn endosperm as a potential drug discovery model for RAW 264.7-Mtb coinfection using histology sectioning with haematoxylin and eosin y staining, optical microscopy with resazurin dye, and real-time cell analysis biosensor assay, an xCELLigence RTCA system assay.

METHODS AND MATERIALS

Scaffold Preparation

Briefly, popcorn endosperm samples were prepared as described above. All plates used in cell viability studies were treated before using pHEMA as previously described unless stated otherwise.

Cell Culture Adherence and Invasion

Confluent (90%) HeLa cells (n + P139) and RAW 264.7 (n + P18) cells obtained from lab stocks were cultured in a T175 flask in basal medium, Dulbecco’s Modified Eagle’s Medium (DMEM) containing 4.5 g/L glucose and supplemented with 10% Foetal Bovine Serum (FBS), 1 % Penicillin-Streptomycin-Amphotericin B (PSA) at 37°C with 5% CO2 in a humified chamber. The cells were sub-cultured using standard trypsinisation protocols, centrifuged, enumerated using trypan blue and a haemocytometer and resuspended in basal medium. All popcorn endosperms were UV sterilised for 1 hr 30 minutes. Once sterilised, the endosperms were transferred into a 6-well or 24-well plate in a Biosafety Class I hood, immersed in basal medium and then initially seeded with cells at a cell density of 1 x 10⁴ per well. Following optimisation, cells were seeded with 5 million cells per well unless stated otherwise — negative controls comprised of wells with media only and popcorn only without any cell seeding. Positive controls comprised of wells with cells only in basal media. Before SEM analysis, plates were incubated for 2 hours at 37°C with 5% CO2 in a humified chamber.

M. tuberculosis H37Rv ApanCD AleuCD derived auxotroph strain mc26206 strain obtained from lab stocks was transformed with GFP plasmid (pUS252) and cultured in 7H9 medium (10% Middlebrook OADC, 0.02% tyloxapol, 24 pg/ml D-pantothenic acid, 50 pg/ml L- leucine and 50 pg/ml hygromycin) in a T25 ml flask at 37°C in a humidified incubator. The culture was maintained by measuring OD600 between 0.6 and 1 .0, keeping the culture in the log phase for infection. /Wtb-GFP was centrifuged at 4 000 x g for 5 min in a swinging bucket centrifuge using the conversion of OD600 1 .0= 3 x 10⁸ bacteria.

For macrophage infection, an MOI of 10 was used. /Wfb-GFP was cultured and centrifuged as mentioned above. The culture was resuspended in basal media, DMEM and added to RAW 264.7 macrophages for four hours for bacteria uptake. This infection cocktail was then placed into a 96-well plate for xCELLigence quantification and onto the popcorn scaffold for histology analysis. The basal medium was removed, washed with PBS and fresh medium replaced for incubation for 12 hours at 37°C with 5% CO2 in a humified chamber before further analysis.

Sterility Analysis

The popcorn scaffold was prepared and seeded as mentioned in above, and then incubated at 37°C in a humified incubator with 5% CO2 for 12 hours in basal medium Dulbecco’s Modified Eagle’s Medium without antibiotic. After 12 hours, 100 pl of media from the incubation was aseptically spread-plated in Nutrient Agar and McConkey Agar plates and incubated at 37°C for 24 hours. Nutrient/MacConkey agar plates were used as sham/negative controls. Colonies present on the plates were counted, and the CFU/ml (Colony-Forming Units) were calculated as follows:

CFU/ml =number of colonies counted x dilution factor x standard volume, 2

Ultrastructural Analysis

The popcorn scaffold was prepared and seeded as mentioned above, and then incubated at 37°C in a humified incubator with 5% CO2 for 12 hours in basal medium, Dulbecco’s Modified Eagles Medium (DMEM) + GlutaMAX containing 4.5 g/L glucose supplemented with 10% Foetal Bovine Serum and 1 % Penicillin-Streptomycin and Amphotericin B. The popcorn endosperm was prepared for SEM analysis as described above.

Micro-Computed Tomography Analysis

The popcorn endosperm scaffold was prepared and seeded as described above and the samples prepared for micro-CT scanning as described above. Sectioning and Histology Analysis

The popcorn endosperm was prepared as stated above. After the 12 hours of incubation, the popcorn endosperms were dehydrated according to Table 3 below.

Table 3: Dehydration of popcorn endosperm

The paraffin Paraplast (Sigma) was melted as per manufacture instructions in an oven (56- 57°C) overnight and remained in the oven until embedding. Moulds for the embedding were made using the wax. After the dehydration, the endosperms were placed on the block moulds, the wax was poured on the samples for 24 hours for the embedding, and the sectioning face was faced upwards. Excess wax was cut away from the sample, and the block was mounted onto a Leica RM2035 rotary microtome. The top layer of the wax was cut off by 15-20 pm, and a 7 pm sectioning size was chosen. After that, the sample was sectioned, and ribbons were created. To reduce the cross-section wrinkling, the sectioned ribbons were placed on a warm water surface (water bath below the wax 40°C). A labelled microscope slide (StarFrost, 26 x 76 x 1.0 mm in dimension) was used to lift the crosssection from the water surface onto the slide. The slide was left to dry in an oven for 48 hours at 37° C.

The following solutions were used in the staining: Acid alcohol solution (0.25 ml HCI, 100 ml ethanol), alkaline water (2 ml ammonium hydroxide, 1 000 ml distilled water), and Haematoxylin solution (Sigma) were used. For the Eosin Y staining, a working solution (250 ml Eosin Y stock solution, 750 ml 80% ethanol, 5 ml acetic acid) was made up from the stock solution (10 g Eosin Y, 200 ml distilled water, 800 ml 95% ethanol) and used in the staining. After that, the slides were stained as follows: Table 4: The Haematoxylin and Eosin Y staining, rehydration, and dehydration of the samples after sectioning

The slides' specimen was preserved with Entellan non-aqueous mounting medium and square coverslips (Marienfeld, 16 x 16 mm in dimension) and visualised using EVOS FL Auto 2 20, Brightfield filter at magnification 10 X, 20 X, 60 X and 100 X for 20 minutes per slide.

Optical Microscopy Analysis

Popcorn endosperms were prepared and seeded as previously mentioned, then stained with phalloidin 594 stain as reported above. Following the staining, the endosperms were visualised on the EVOS FL Auto 2 using the Brightfield, RF Red filters at 10 X magnification. Three independent measurements of each experiment were made, and the images were collated using Inkscape software (Version (Ver.) 1.1 ; https://imagej.nih.gov/ij/download.html).

Cell Viability Assays

The popcorn endosperm was prepared as stated above. After seeding and incubation, 20 pl of resazurin (1.5 mg/ml) was added to the popcorn endosperms for four hours of incubation. Controls involved were negative controls with just media, popcorn only without seeding and cells seeded in wells. After that, the media was removed from the wells, put in new plates, and measured the fluorescence using 560 nm excitation/590 nm emission. The Biotek Synergy Mx plate reader with Gen 5.1.10 software was used to read the fluorescence. The cells were taken from the well, and the fluorescence was read without any additional processing.

Long-term Cell Culture

Approximately 2 x 10⁶ MDA-MB-231 cells were seeded in non-adherent 6 well plate on popcorn endosperm that was fragmented in 1 cm sections and incubated for 48 hours in DMEM High Glucose with 10% FBS containing antibiotic-antimycotic solution to allow swelling. Cells were seeded and allowed to incubate in static conditions for 14 and 30 days. At appropriate time points wells were selected for staining using ReadyProbes Cell Viability Imaging Kit Blue/Green (Thermo). Cells were observed on EVOS FL Auto 2 after 30 minute incubation with viability stain. To further assess the ability of the scaffold to support mammalian cells of a different phenotype, the scaffold was prepared using a commercial hotair popper as opposed to a standard microwave popper. Endosperms were separated and prepared as before and incubated in the presence of ~1 million C2C12 mouse myoblast cells in adherent and non-adherent conditions in DMEM (high glucose) supplemented with 10% FBS and 1 % antibiotic-antimycotic solution. Control wells contained no cells and endosperm (negative) or cells only (positive). Cells were allowed to grow in the presence (or absence) of scaffold for 12 days prior to staining with ReadyProbes Cell Viability Imaging Kit Blue/Green (Thermo). xCELLigence Analysis RAW 264.7 cells were infected as described above, and the co-culture was seeded into a 96-well xCELLigence E-plate. The e-plate has gold microelectrodes at the bottom that calculates the cell index (Cl) by measuring changes in electrical impendence from each well at a specific time compared to an initial background impedance. This plate was used to calculate the cell index using the electrical impedance released by each well at 30- minute intervals. This data was then recorded by the xCELLigence RTCA software, which then recorded the cell index at various time points. The cell proliferation of infected RAW 264.7 cells over 72 hours before infection, during, and after infection was tracked and measured and the xCELLigence RTCA software and Excel were used to generate representative graphs.

CI= Z-Z_o3O ... ,3

Cl is the cell index, Zi is the electrical impendence from a well at a specific time, Z0 is the background impedance, and 30 minutes is the time interval between measurements.

Data Analysis

The data obtained from the three independent experiments (plates) and three independent popcorn endosperm samples were analysed to determine the statistical differences between the various samples and times mentioned above. The one-way ANOVA (analysis of variance) test was used, and a p-value of < 0.05 was considered significant. Microsoft (MS) Excel software and TIBCO Statistica (14.0) were used to generate graphs and calculate the significance.

RESULTS

Sterility Analysis

The sterility of the popcorn endosperm after preparation and expansion was investigated after incubation in basal medium for 24 hours at 37°C in a humidified incubator with 5% CO2. The medium was plated on Nutrient and MacConkey Agar plates at 37°C for 24 hours, following UV light sterilisation for 1 hr and 30 mins to determine the efficacy of UV sterilisation on the popcorn endosperm. Colonies present on the plates were counted, and images of the plates were taken. These images were collated using Inkscape software. The CFU/ml (colony forming units/ml) was calculated and summarised in Table 5. The nutrient agar plates and MacConkey agar plates were used. The control plates, which had no spread plating, were compared to two tests plates of both agars, which were spread plated. The colonies were then counted, and the efficacy of the sterilisation method is represented quantitatively in Table 5, which shows the colony-forming units on both types of plates.

Table 5: Sterility studies of the popcorn endosperms following UV light sterilisation for 1 hr 30 min.

The sterility of the popcorn endosperm needs to be tested for applications in tissue engineering. Sterility is an essential factor for biomaterial selection as the contamination of the biomaterial would result in an immune response being elicited, rejection of the implanted cell-seeded scaffold or loss of function. The negative control had no colony growth. This demonstrated that the agar used then (Vegitone agar) itself had not been contaminated and validated the experiment results. The unsterilized popcorn showed a colony spread that was too many to count. UV light sterilisation has been proven to work as a sterilization method but may result in the surface modification of polymers.

The colonies on both the MA and NA plates were small, white, and round. In MA plates, white colonies are non-lactose fermenters and could be bacteria such as Pseudomonas and Salmonella, of which the latter is usually associated with food contamination. While these results showed colonies formed on the plates, this was an effective method of sterilisation. Research has shown Salmonella contamination post microwave expansion of popcorn and points to a need for surface sterilisation and other forms of sterilisation before expansion. Additionally, post microwave expansion, the removal of husks was performed outside a biosafety cabinet before UV sterilisation, increasing the likelihood of microbial contamination.

Other sterilisation methods, such as sterilisation with dry heat and gamma irradiation, could be considered. This shows that antibiotics are crucial and will assist in adding to the sterility of the biomaterial scaffold in vitro, especially when cell-seeding.

Ultrastructural Analysis

The ultrastructure of the popcorn endosperm was investigated after the microwave expansion of the popcorn endosperm, cell seeding with HeLa and RAW 264.7 cells and incubation in cell media for 12 hours. The samples were fixed with glutaraldehyde overnight, dehydrated with increasing ethanol concentrations, dried with a critical point drier, coated with gold, and then analysed using the TESCAN Vega TS 5136LM scanning electron microscope. Micrographs were recorded at an acceleration voltage of 20 kV and analysed using Vega TC and Inkscape software. The results show the preliminary cell seeding with HeLa cells at 1 x 10⁴ cells per endosperm. Cell-like structures were observed on the scaffold (data not shown). These cell-like structures were observed on multiple independent popcorn endosperms. To determine if this phenomenon was observed with other cell lines, the experiment was repeated with RAW 264.7 cells. The result of this experiment, as with the HeLa cells, showed multiple cell-like structures on multiple independent popcorn endosperm samples.

The size of HeLa cells is approximately 20-40 pm in diameter, and this correlates with the size and scale of the cell structures on the SEM images and of RAW 264.7 cells to be approximately 20 pm in diameter. When comparing the size of these cells to the size of the popcorn endosperm ultrastructural pockets, the cells can infiltrate and proliferate on the ultrastructural pores, which are about 40 pm large in diameter on average. Additionally, these cell-like structures are clumped together, which is the typical behaviour of these cells and is known to improve proliferation due to cell signalling and cytokines. This shows the popcorn endosperm to be a useful biomaterial used for tissue engineering applications for soft tissues as the cells are growing on the ultrastructure and appear to be biocompatible with the popcorn endosperm and the porous microstructure. This also points to the bioactivity and biocompatibility of the scaffold. Micro CT Analysis

The surface structure of the popcorn endosperm was investigated after microwave expansion, cell seeding with HeLa and RAW 264.7 cells and incubation in cell media for 12 hours. The samples were then immersed in PBS for an additional 24 hours for transporting to the micro CT facilities and then analysed using the Bruker SkyScan 1173 micro-computed tomography scanning system. Three-dimensional scans were analysed and constructed using CTVox software. The macropores visible in the initial micro-CT scan without cells was still able to be observed. Cell-seeded endosperms viewed at different angles, including the frontal, bottom, and side show that the pores run through the entire endosperm and not just on the surface. Additionally, various sliced images display the microporous structure of the endosperm that has been maintained and is still intact post- cell-seeding. There are grooves and ridges as well as pores observed in these seeded popcorn endosperms. It was observed that the seeding of cells did not disrupt the surface structure of the popcorn endosperm as it appears to be similar to the non-seeded cells. These characteristics are ideal for biomaterials used in tissue engineering applications and point to the bioactivity, biocompatibility, and mechanical and structural integrity of the popcorn endosperm scaffold.

Histology Analysis

The popcorn endosperm's cell adherence and infiltration capabilities were assessed after the microwave expansion, seeding with HeLa and RAW 264.7 cells and incubation in cell media for 12 hours. The samples were dehydrated, embedded with wax, sliced with a microtome, and then stained with haematoxylin and eosin Y. The specimen slides were then analysed using the EVOS FL Auto 2, Brightf ield at magnification 10 X, 20 X, 40 X and 60 X. The images were collated using Inkscape. The polygonal pockets are still visible, and appear intact and structurally rigid despite the dehydration and sectioning treatments. These sectioned slices are estimated to be at a depth of 126 pm. The incidence of the cells was enumerated to provide the efficacy and depth of the cell infiltration into the scaffold and its bioactivity.

Microtome sectioning of the popcorn endosperm without cell seeding showed the polygonal ultrastructure of the popcorn endosperm. This aligns with the SEM images that also display the polygonal structure. It was also shown that the polygonal structures run deep into the popcorn endosperm and that it is not just located at the surface of the popcorn. Additionally, most of the structure appears to be intact. Popcorn endosperm samples seeded with HeLa cells also showed the polygonal structures. Studies have shown ECM deposits of HeLa cells as soon as 24 hours post-cell seeding. This was accompanied by the spreading of the cells induced by adherence to the substrate it had been seeded onto. Further testing showed the ECM deposits increased as the days of infiltration of HeLa cells into the scaffolds increased, and cells appeared less distinguishable as they deposed into the ECM. This is in accordance with the results of this study, which show a depth infiltration of at least 126 pm. These cell-like structures appear to have infiltrated into the popcorn endosperm pockets. As the slices were cut approximately 7 pm thin, the cells did not just adhere to the surface but appeared to have infiltrated the scaffold. The size of the cells is 50 pm, with the average calculated size of the popcorn endosperm pockets arranged at 36-40 pm. As HeLa cells are approximately 20-40 pm and RAW 264.7 cells are approximately 20 pm, these size comparisons show that the cells can fit and infiltrate into the polygonal pockets of the popcorn endosperm.

Haematoxylin and Eosin Y were the stains used in this histology analysis, and they stained cell nuclei blue and Eosin Y, a counterstain stains cytoplasm and the ECM pink. RAW 264.7 cells are smaller than HeLa cells but appear to have infiltrated the endosperm. Studies have shown RAW 264.7 cells to adhere and spread onto scaffolds they are seeded onto within 24 to 72 hours of seeding as they produce extracellular proteins while adhering to the scaffold. The adherence of RAW 264.7 cells onto biomaterial scaffolds also increased in highly porous scaffolds compared to low porosity scaffolds. This confirms the biocompatibility of the popcorn endosperm, leaving the viability of the seeded cells in the endosperm to be analysed.

For the HeLa cell-seeded endosperm, in approximately 28 pockets at an estimated depth of 126 pm, seven cells were observed. Of these seven cells, five were individual cells and a potentially observed pair in a state of cell division. A similar phenomenon was observed in a second HeLa cell-seeded endosperm studied, wherein approximately 40 pockets at an estimated depth of 315 pm, eight cells were observed, of which three pairs were also splitting from each other. For the RAW 264.7 cell-seeded endosperm, at an estimated 38 pockets at a depth of 161 pm, seven cells were observed. All these seven cells were individual cells. In another RAW 264.7 cell-seeded endosperm studied, in 37 pockets at an estimated depth of 287 pm, seven cells were observed, of which four cells were splitting from each other. The other three cells were individual cells. The levels of incidence between the various depths appear to be the same. There was no significant difference between the cells counted at a depth of 161 pm and 287 pm.

Optical Microscopy Analysis

The preparation of the popcorn scaffold was performed as previously reported. The samples were incubated at 37°C in a humified incubator with 5% CO2 for 12 hours in basal medium after cell seeding with HeLa cells and RAW 264.7 cells. The popcorn endosperms were fixed with PFA at room temperature and then stained with phalloidin 594 to visualise the cells using the EVOS FL Auto 2 microscopes at 10 x magnification. Results showed that the cells are either in the scaffold or on its surface. Phalloidin staining is used to stain f-actin, a protein found in eukaryotic cells. This stain was used on the popcorn endosperm for optical visualisation of the scaffold and the cells. Fluorescence signals of the same cell shape as the control images were observed on the popcorn scaffold. This phenomenon was seen with both HeLa and RAW 264.7 scaffolds. Images of the merged brightfield and fluorescent views showed the location of these cells within the scaffold and confirmed that the cells are infiltrating and invading the scaffold. These results point to the appeal of the popcorn endosperm to be a biomaterial used in both soft and hard tissue engineering applications.

Cell Viability Analysis

The cell viability of HeLa and RAW 264.7 cells was analysed after the microwave expansion of the popcorn endosperm, seeding with HeLa and RAW 264.7 cells, and an infection analysis with RAW 264.7 cells and Mtb. A separate analysis was done without seeding onto the popcorn endosperm for control testing. After seeding and incubation for 12 hours, resazurin was added, and the fluorescence (560, 590) was read after four hours using the Biotek Synergy Mx plate reader with Gen 5.1.10 software. The analysis of the resazurin to resorufin change was compared. Due to the use of pHEMA plates in all experiments, a control experiment was performed to test the viability of the cells seeded in adherent tissue culture plates and rule out the interference of the resazurin signal read by popcorn endosperm particulates. The cells were adherent to the plate and metabolically active, showing high resazurin to resorufin signal reads. The results of the resazurin to resorufin conversion by cells seeded in popcorn endosperms was observed. In this analysis, high resazurin to resorufin conversion signals show metabolically active cells after seeding onto popcorn endosperm for more than 12 hours.

The popcorn endosperms seeded with HeLa cells had a fluorescence read, which was more than two times higher than the control. All three different samples of popcorn endosperm were shown to have a high fluorescence signal of more than 2 000 RFU. Additionally, the control of popcorn only shows that the high signal observed in the seeded popcorn was due to viable and metabolically active cells converting resazurin to resorufin as the media control of the popcorn only had a low resazurin signal, similar to that of the media only control. This same pattern was observed for the RAW 264.7 cells.

Cells were metabolically active and showed that the cell-seeded endosperms were metabolically active and viable, proving the popcorn endosperm is biocompatible (See Fig. 5). The ability of the popcorn endosperm to support cell infiltration and invasion of metabolically active cells, as well as a potential infection analysis 3D model, further confirm the bioactive and biocompatible nature of the popcorn endosperm and its ability to be an ideal biomaterial for tissue engineering applications.

Long-term Cell Culture

Non-adherent culture vessels force cellular attachment on permissible surfaces. Following the success with the use of polyHEMA treated well plates, a commercial non-adherent/low cell binding plate was used for investigation of long term attachment, proliferation and survival on the endosperm scaffold. Cells were cultured in static and fed-batch conditions, i.e. fresh media was diluted into the culture every 3-4 days. In both cases cells were observed to grow on the surface of the scaffold for periods longer than 10 days. The MDA- MB-231 cell cultures were monitored over 30 days and the C2C12 cells were cultured up to 12 days. The open pore structure likely facilitates nutrient and gas exchange allowing long term mammalian cell infiltration, colonization, and survival. Cell survival will likely be improved in dynamic culture conditions. Interestingly, there was a greater relative amount of live cells observed relative to dead cells for the C2C12 model this may be as a result of the fed-batch approach to cultivation, i.e. 1 mL fresh media was fed into the culture every 3 to 4 days. The experiment was performed over a shorter time period relative to the MDA-MB231 cell culture. The scaffold only control panel it was apparent that the live/dead stain bound to the starch backbone (live) and uncharacterized foci (dead), this is unlikely to be plant cell nuclei. An alternative stain may be required. Relative to the cell only control, the scaffold appears to provide an adequate surface for cell adhesion, proliferation and survival (C2C12 + Scaffold). Longer term survival and formation of fused myofibres were not observed as cells were not exposed to differentiation conditions.

It should be noted that all these experiments were performed in largely static cultures and further optimisation is required for gas and nutrient exchange to use the scaffold in dynamic culture environments, i.e. bioreactors. Further surface modification should be considered, e.g. collagen treatment or pre-enzymatic digestion.

Validation of Cell Infection Model

The popcorn endosperm was validated for drug discovery applications in tissue engineering through cell infection models and analysis. To validate the efficacy of the infection model without the popcorn endosperm scaffold, RAW 264.7 cells were cultured as previously reported and infected with Mtb for 12 hours. After that, the infected cells were stained with auramine-rhodamine and then visualised. Real-time analysis of the infection of the RAW 264.7 cells was performed using the xCELLigence RTCA e-plates and software. This is shown in the cell index vs time curve observed in Figure 4. The RAW 264.7 cells were infected and seeded into the plates, and the cell index of the infection was analysed over 17 hours. Sham controls consisted of wells in the e-plates seeded with DMEM, RAW 264.7 only cells, and Mtb only cells to offer a comparative reference for the infection. The experiment consisted of three independent replicates that were averaged and graphed in Excel to show the progression of the cell indices over the 17 hours. This offers a qualitative measure and validation of the infection model before testing it onto the popcorn endosperm scaffold. The popcorn endosperm was prepared and seeded with Mtb infected RAW 264.7 cells for 12 hours. After that, the scaffold was sectioned at an estimated depth of 175 pm and 280 pm into the scaffold and stained with haematoxylin and eosin for visualisation. Microscopy analysis was done using the EVOS FL Auto 2 microscope, and all images were collated using Inkscape software.

Drug discovery and infection models that are 3D and physiologically accurate, especially for infectious diseases like TB, are vital. As highlighted previously, there is a need for innovative drug discovery models that possess the ideal characteristics of a biomaterial scaffold. Following the characterisation and validation of the popcorn endosperm as a potential biomaterial scaffold for tissue engineering applications, it was imperative to validate an infection model for one of the most infectious diseases plaguing South Africa.

Infection of RAW 264.7 cells by Mtb was demonstrated. The brightfield view of the infected cells with Mtb. and Auramine-Rhodamine staining used to identify acid-fast bacteria such as Mtb show the Mtb stained cells in bright yellow. This shows that the RAW 264.7 cells are being infected by Mtb. This qualitative validation aligned with the quantitative results observed in Figure 4.

In Figure 4, the cell index vs time curve is shown for the RAW 264.7 and Mtb infection. This was performed using the xCELLigence RTCA system, which uses gold microelectrode electric plates to read signals emitted by metabolically active cells. Cell behaviour such as adherence, cytotoxicity and signalling can be measured using this system and analysed (Hamidi et al., 2017). This gives much more information on detailed signals emitted by cells at various time points of the cell cycle than a cell viability assay like resazurin, which does not provide such detailing. The sham control was performed with just DMEM basal medium and had cell index readings below 0, which was expected for the negative control, which had no cell seeding meaning the e-plates were unable to read any signal as there were no cells emitting signals. This showed that the xCELLigence system was functioning as expected. RAW 264.7 cells and Mtb cells were seeded onto the plate for controls to ensure that the cells were metabolically active and independent of each other. The RAW 264.7 only cells had the highest cell index as observed in the time curve compared to the Mtb only control, which also had a high cell index but not as high as the RAW 264.7 cells. As the RAW 264.7 cells are more prominent than Mtb, a higher cell index was expected when seeded into the e-plate. Additionally, RAW 264.7 cells are adherent cells and would naturally adhere to the e-plate, whereas Mtb in culture grows in strings, lowering its adherence to the e-plate and, as a result, a signal detected by the xCELLigence system. The infection of the RAW 264.7 cells by Mtb also showed a high cell index rate in comparison to the control, as expected. However, the cell index curve was lower than the controls, with only RAW 264.7 and Mtb cells. As the cells were being infected, a lower cell index was expected as the RAW 264.7 cells would result in reduced proliferation due to the pathogen being introduced to its environment and its conversion to macrophage cells to engulf the Mtb pathogen. This was the second validation of the infection model. Following this, the infection model was then tested on the popcorn endosperm, and histological analysis was performed to observe whether the popcorn endosperm could be used as a potential drug discovery and research model for another use in tissue engineering and drug discovery applications.

RAW 264.7 cells infected with Mtb seeded into the popcorn endosperm scaffold followed by sectioning and histological analysis was performed and showed polygonal structures similar to those previously seen, and their structural integrity was still maintained. This observation is favourable as it means the addition of Mtb has not visibly affected the scaffold. Further magnification until the nuclei of the cells that have infiltrated the scaffold were visible showed that in approximately 40 pockets at an estimated depth of 175 pm, eight cells were observed. Of these eight cells, two looked to be individual cells, while the other six were from three pairs of cells close together. A similar phenomenon was observed at an estimated depth of 280 pm, where nine cells were observed, of which three pairs were also splitting from each other.

With reference to Figure 6, there is provided a graph pertaining to nucleic acid isolation from scaffold where Lane 1 band 2 was used as reference band for relative quantification of DNA within the highlighted region of interest (white rectangle) in (A). (B) shows the relative quantities of isolated DNA. With reference to Figure 7, the bar graphs show the resazurin fluorescence (Ex 530 nm, Em 590 nm) readings after incubation of corn endosperm scaffold with or without C2C12 cells in adherent and/or poly-HEMA coated plates. Cells were seeded on com endosperms at O, 5000 (5K), 10000 (10K), 20000, 30000 (30K), 40000 (40K), 50000 (50K), 60000 (60K), 80000 (80K) and 10000 (100K) cells per well for (A) 4, (B&C) 7 and (D) 21 days. In Figure 6C GM stands for growth media; S for scaffold; CO stands for cells only; Adh stands for adherent plate; PH stands for poly-HEMA coated plate and C cells. Multiple samples (n>6) were used. Error bars represent standard deviation.

With reference to Figure 8, SEM micrographs are provided of the ultrastructure of the popcorn endosperm without cells and following seeding with C2C12 cells and SEM sample processing for 21 days. Scaffold only: The popcorn endosperm showing polygonal pockets of dry scaffold with cell-like structures attached to the surface indicated by the white arrows. Magnification range from 479 to 7100 x (A-D).

CONCLUSION

HeLa, RAW 264.7, MDA-MB-231 and C2C12 cells are well-established and researched cell models for testing various applications in biology. One of these applications includes scaffold and biomaterial testing. This is one of the reasons why HeLa and RAW 264.7 cells were used in the preliminary biocompatibility testing for the popcorn endosperm.

SEM images from seeding the popcorn endosperm with HeLa and RAW 264.7 cells showed rounded, elongated, and flattened structures observed in clumps, consistent with the size and shape of Hela and RAW 264.7 cells at various cell cycle stages (results not shown). These structures are not observed in the control SEM images. It was demonstrated that inner polygonal pockets include cells invading the processed scaffold and this points to the mechanical strength and structural integrity of the popcorn endosperm. This also shows the depth into which the cells were seeded and embedded into the endosperm and not only on the surface. The observation of cell-like structures in all the SEM images shows the seeded cells attached to the ultrastructure. The micro-CT scans performed confirmed that the seeding of the two cell lines, Hela and RAW 264.7 cells, did not cause any alterations or disruptions to the endosperm structure. This shows the potential of the popcorn endosperm to be biocompatible with cells. Furthermore, the porosity of the scaffold material indicates the feasibility of gas and nutrient exchange.

The microtome sliced and histologically treated and stained sections of the popcorn endosperm images showed that the cell attachment and infiltration into the popcorn endosperm are not only on the surface of the endosperm structure but also at least 129- 315 pm deep into the scaffold itself. Both in the HeLa and the RAW 264.7 cell seeded endosperms were observed. Cells were shown to be embedded into the polygonal pockets of the popcorn endosperm. This was further validated by the phalloidin stained images, where HeLa and RAW 264.7 cells in the popcorn endosperm scaffold were stained red. As phalloidin binds f-actin in eukaryotic cells, it can then be concluded that the cell infiltration of the popcorn endosperm is a consistent and reoccurring observation.

With these images histological and optical microscopy images confirming the bioactivity and biocompatibility of the popcorn endosperm as an ideal biomaterial scaffold, it was left to observe whether or not these attaching to and infiltrating into these polygonal pockets are metabolically active and viable as it is possible that while they may be embedding into the polygonal pockets, they may not be bioactive, which would disqualify the popcorn endosperm as a potential to be a scaffold for tissue engineering applications. Resazurin, a commonly used, non-toxic and inexpensive phenoxazine dye, was used to test the viability of the cells seeded into the popcorn endosperm scaffold. The cells were metabolically active and viable as they showed a high fluorescence output, which was statistically significant from the control (p < 0.5). To ensure that the plate reader picked up actual metabolic cells and not background signals from the popcorn endosperm being in the tissue culture wells, only the seeded endosperms were read. The media showed high fluorescence read across the board and were significantly higher than those of the controls, showing that the cells that were seeded into the popcorn endosperms were not only viable but also metabolically active and converted resazurin to resorufin. This validated and confirmed biocompatibility characteristics with various cell lines. An infection model study was formulated to observe whether the popcorn endosperm scaffold can be used in other tissue engineering applications such as drug discovery models. RAW 264.7 cells were first infected with Mtb and stained with auramine-rhodamine to determine that the 2D infection model was viable and worked before infection with the 3D model. Mtb was stained yellow, confirming the infection of the RAW 264.7 cells by Mtb. To further validate this before testing onto the popcorn scaffold, a real-time analysis of the cell index against the time of the progress of infection of RAW 264.7 cells by Mtb was performed using the xCELLigence RTCA system. This showed that the infection of the RAW 264.7 cells was ongoing as, over time, the cell index increased but, in comparison to uninfected RAW 264.7 cells, had a lower cell index highlighting that there was a reduction in proliferation rates as time increased. Following this validation, RAW 264.7 cells were infected with Mtb, and the infected cells were seeded onto popcorn endosperm. This histological sectioning was made, stained with haematoxylin and eosin y and visualised. Infiltration of the infected RAW 264.7 cells into the scaffold of at least 280 pm was observable. This shows the potential of the popcorn endosperm not only to be a novel, inexpensive, easily sourced, structurally sound, bioactive, and biocompatible biomaterial for tissue engineering applications such as soft and hard tissue formation but drug discovery and research models for infectious diseases such as Mtb.

Further analysis of cellular growth and survival over a prolonged period using human MDA- MB-231 breast cancer cells and immortalised mouse C2C12 skeletal muscle cells show the potential of the scaffold to support long term cell culture in batch and fed-batch conditions. The potential of expansion to a bioreactor based dynamic culture system expands the use of the scaffold material in particular with respect to use in culturing synthetic meat for cellular agriculture.

A summary of the characterisation of the popcorn endosperm as a biomaterial is shown in Table 6 below. The studies show the popcorn endosperm to be mechanically and a structurally sound, hydrophilic, biocompatible, and biodegradable scaffold for tissue engineering and regenerative medicine applications.

Table 6: Summary of the ideal characteristics of a scaffold tested for on the popcorn endosperm

Claims

1. A method for preparing a porous three-dimensional bioscaffold from corn kernel endosperm, including translucent com kernel endosperm, comprising or consisting of the steps of: a) providing a plurality of com kernels; b) exposing the com kernels to a dry heat source, including a popcorn maker or microwave source without addition of oil for a period of time suitable to result in a cooked and expanded endosperm and a husk; c) removing the pericarp or husk from the endosperm and discarding it; and d) sterilising the endosperm including by Ultra-Violet (UV) sterilisation, dry heat sterilisation or gamma radiation thereby to form a porous three-dimensional com kernel endosperm bioscaffold.

2. The method according to claim 1 , wherein the com kernels are Zea mays kernels, including Zea mays L var. everta kernels which form butterfly popcorn endosperm once expanded.

3. A method for culturing living cells bound to and within a porous three-dimensional com kernel endosperm bioscaffold prepared according to the method of either claim 1 or claim 2 comprising the following steps:

(iii) incubating the cell culture and bioscaffold, thereby to allow the to cells grow and infiltrate the porous bioscaffold such that the living cells are bound to and within the bioscaffold.

4. A porous three-dimensional corn kernel endosperm bioscaffold prepared according to either claim 1 or claim 2, which comprises living cells, including mammalian, avian, fish or invertebrate cells bound to and within the bioscaffold.

5. An artificial tissue comprising the porous three-dimensional com kernel endosperm bioscaffold prepared according to any one of claims 1 to 3 which comprises living cells, including mammalian, avian, fish or invertebrate cells bound to and within the bioscaffold.

6. The artificial tissue according to claim 5, wherein the artificial tissue is comprised within a synthetic meat product.

7. A method for drug or biological medicament discovery, drug or biological medicament resistance assessment and/or drug or biological medicament cytotoxicity assessment with the use of the porous three-dimensional corn kernel endosperm bioscaffold prepared according to any one of claims 1 to 3 which comprises living cells, including mammalian, avian, fish or invertebrate cells bound to and within the bioscaffold.

8. The method according to claim 7, wherein the mammalian cells are human cells.

9. The method according to either claim 7 or 8, wherein the method further includes a step of treatment of the living cells with one or more compound(s) to assess the suitability of such compound(s) as a drug or biological medicament for treatment of a subject.

10. The method according to either claim 7 or 8, wherein the method further includes a step of infecting the living cells bound to and within the bioscaffold with a pathogen including a bacterial, viral or fungal pathogen, followed by treatment of the infected living cells with one or more compound(s) to assess the suitability of such compounds to inhibit or kill the pathogen without harming the living cells.

11. An ex vivo or in vitro diagnostic method comprising the use of the porous three- dimensional com kernel endosperm bioscaffold prepared according to any one of claims 1 to 3, or an artificial tissue comprising the porous three-dimensional corn kernel endosperm bioscaffold.

12. A method of culturing living cells including mammalian, avian, fish or invertebrate cells in a bioreactor with the use of the porous three-dimensional com kernel endosperm bioscaffold prepared according to any one of claims 1 to 3.