plant bioprinting and its uses in agriculture

printing live plant cells and its uses in agriculture

1. Introduction Why Plant bioprinting ? So WORLD HUNGER will be the huge crisis in future Malnutrition, unhealthy diet, starvation – reasons for hunger Reducing arable land, hence growing food in laboratory is one of options TO MEET THE DEMAND. TRADITIONAL ESTIMATION OF world population reaching 9.2 billion. (Quantity is achieved but quality havn’t achieved hence, nutritional specific bioprinted food can be made using this technology. Hence, lot of smart people are finding ways to meet the demand. Hence, focusing on sustainable future, Bioprinting of food is one of the options to meet quality food demands To give the gest of my presentation. Take a plant cell and supporting material, mix together to form a bio ink Put in a printer Get a 3D construct having live cells Induce live cells in 3D construct with Magic factors (Growth factors and differentiation factor)to differentiate into desired economic part

2. Vimanth S II Ph.D. ( MBB ) UNIVERSITY OF AGRICULTURAL SCIENCES, BANGALORE ADVANCED CENTER FOR PLANT BIOTECHNOLOGY Seminar on Novel Biotechnological approaches in Plant Science using Plant Bioprinting Course: MBB 681 (0+1)

3. additive manufacturing, commonly known as three-dimensional (3D) printing. Used for tissue engineering and regenerative medicine, where complex structures of living cells and biocompatible materials were shaped into functional tissues and organs. it allows the fabrication of objects by the addition (or deposition) of material layer-by-layer to create an object with the desired shape. The material deposition or fusion is controlled by 3D printer equipment’s based on a 3D in silico model generated by a computer aided design (CAD). Bioprinting is a technique to develop multidimensional biological products with natural functionality using multidisciplinary approaches of computational designing, systems biology, and material science. Definition of Bioprinting

4. Why plants are subjected to Bioprinting ? plant cells are totipotent, that is, they have a strong potential to develop a tissue scaffold that acts as the precursor for an organ. From that organ, an entire plant can be generated, even if the environmental conditions are unfavorable. To harness following Alkaloids, polysaccharides, glycosides, terpenoid lactones, and flavonoids are important phytochemicals that have been linked to plant immunomodulation activities In vitro synthesis from plant cell cultures could meet the growing demand for plant-derived components, such as secondary metabolites, in medicine, and cosmetic industries.

5. History of Green Bioprinting : 1979 by Peter Brodelius and his team , immobilizing biocatalysts (enzymes, microbes, etc.) for plant cell culture. immobilization presents advantages in the biotransformation of compounds. immobilized plant cells were shown to be able to proliferate and to be biosynthetically active, leading to an increased production of secondary metabolites, when compared to cells suspension. 1994 study by Vanek et al., method of 131 immobilization influenced the biotransformation path of the cis-verbenol, application in essential oils production, entrapping Solanum aviculare cells in formulations containing alginate, pectin or carrageenan. cis-verbenol conversion into trans-verbenol or verbenone. 2015 by M. Gelinsky and his team, bioprinting technique which uses extrusion-based method to cultivate green algae in a spatially organized manner. Chlamydomonas reinhardtii microalgae growth over 12 days within their alginate/methylcellulose bioink. proliferation was confirmed by microscopy imaging. monitoring active photosynthetic metabolism (oxygen release and chlorophyll synthesis). extrude Ocimum basilicum (Basil cells) seeded in their bioink and managed to cultivate the 3D bioprinted structure for 20 days.

6. food manufacturing, Leuven Rega Institute for Medical Research used Valerianella locusta (lettuce leaf cells) to screen for bioink composition based on pectin. College of Life Science and Biotechnology in Korea University implemented the Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting method and managed to grow Daucus carota L carrots cells for 35 days

7. Gillet F 2000 Nicotiana tabacum (Tobacco) cells were encapsulated in an alginate matrix with the aim of increasing the secreted Scopolin bioproduction yield (Plant metabolite) Antiinflammatory,anti oxidant properties Brodelius 1982 Morinda citrifolia (Noni) cells were for example entrapped in alginate microbeads and demonstrated a change in their metabolism while being cultured in 3D environment 2005, Philip Ball Salmon skin grafts gelatin gels Hopfner et al. (2014) demonstrated a first co-cultivation of the microalga C. reinhardtii with murine fibroblasts in direct contact on both 2D tissue culture polystyrene grafts and on 3D collagen-based scaffolds for skin tissue regeneration. The microalga showed high biocompatibility and photosynthetic activity under co-culture conditions (at 30 ◦C and in a 1:1 mix medium)

8. Beckwith et al. on the Zinnia elegans plant model, plant cell differentiation by plant hormones modulation within the 3D construct regulating ratios of auxin and cytokinin hormones. obtain different ratios of xlignification of the constructs. a key step for the in vitro formation of wood and opens the way to the study/control of the structural properties of such cell-filled architectures. increased amount of α-naphthaleneacetic acid (NAA) and 6-Benzylaminopurine (BAP), “plant cell production through the direct growth of specific plant tissue, free of unwanted components, with modifiable properties and controllable architecture” upcoming results of climate change, like the declining land availability, the seasonal and climatic susceptibility, as well as the low yields of useful plant materials and the length of the plants cultivation. Cells isolated from the leaves of Zinnia elegans were mixed with Gellan Gum ( Gelzan CM, 3 g L-1) in a ratio of 1:3 (v/v)

9. Scaffolds : scaffolds are fibrous, porous, or permeable threedimensional (3D) biomaterials that allow biological liquids and gases to pass through while facilitating cell interaction, viability, and extracellular matrix (ECM) deposition with minimal inflammation and toxicity while biodegrading at a controlled rate. Classification of Bioinks : Scaffold based bioinks : combine cells into an exogenous support structure, that aid cell adhesion and growth, provides mechanical strength, Scaffolds are supposed to degrade in time to form desired tissues while cells proliferate. scaffold-free bioinks . produced entirely using cells and their generated matrices, capacity of cells to self-assemble into bigger tissue structures, reduces immunological responses in vivo ie ,mimic the cell microenvironment of native organs, deposition of a high cell density. Properties of scaffolds : Porous Biodegradable Transparent

10. Hydrogels (water-based gels), which are biocompatible and have an extracellular matrix (ECM)-like qualities. modifiable chemical structures biodegradable qualities ability to hold live cells customizable mechanical features ability to generate acceptable resolution during printing immobilization strategies have been used for plant cells, such as encapsulation in hydrogel beads (such as agarose, gelatin, alginate, and pectate) Types of Hydrogels natural biomaterials : promote cell development by mimicking the natural ECM, self-assembling, and enabling biocompatibility synthetic biomaterials : are more structurally manageable and have the potential to photocrosslink ; however, they can also be more cytotoxic than natural material. promising options for modifying features including mechanical qualities, printability, and cross-linking Hybrid Bioinks

11. natural biomaterials: Agarose is a natural polysaccharide obtained from red seaweed that comprises repeating β-D-galactose and 3,6-anhydro-α-L-galactose disaccharide units, thermo-reversible gelling mechanism. 50 mM and 1500 mM ca2+ Alginate is a brown algae-derived anionic polymer composed mainly of blocked copolymers, with α-(1-4)- L-guluronate and (1,4)-linked β -D- mannuronate residues, biomedical applications such as wound healing, drug delivery, and tissue engineering because of their low cost and biocompatibility. Cellulose, a very rigid polysaccharide made up of (1 – 4) linked β-D-glucopyranosyl units linked together, plays are the primary structural substance in plant cells

12. Chitosan: Collagens are the most widespread proteins found in mammals, accounting for roughly 30% of the average total protein mass in mammals, unique number of triple helices and chain combinations Silk fibroin (SF) is a highly versatile natural protein derived from silkworms, mechanical characteristics, biocompatibility, and easily regulated degradability

13. synthetic biomaterials : poly(ethylene glycol) (PEG) are the most often utilized synthetic polymers within the field of 3D bioprinting. Polyethylene (glycol) diacrylate (PEGDA) and poly (ethylene glycol) methacrylate/ dimethacrylate are the most prevalent PEG hydrogels utilized for bioink materials.

14. Gelation Kinetics Newtonian and non-Newtonian Shear stress/ yield stress

15. What is Bio-Ink ? Bioinks are basically biomaterial solutions containing living cells and are essential components in bioprinting. Bioprintability non-toxicity insolubility in cell culture medium visco -elasticity high mechanical integrity and stability the ability to stimulate cell adhesion biodegradability at a steady rate are all necessary features of biomaterials for enabling high-quality tissue regeneration. Non-immunogenicity and permeability of nutrients and gases are also critical characteristics of an ideal bioink

16. Two Characteristics of Bioink: Shear thinning is a rheological behavior which consist in decreasing viscosity of the bioink when shear stress is applied. This allows to protect cells from cell lysis while being extruded yield stress which represent the shear at which bioink start to be fluidized. Properties of Bioinks : Visco -elastics Non-toxic Good mechanical strength Structural integrity once bioprinted Biocompatibility

17. Parts of 3D bioprinter Nozzle (Bioink dispenser) (Temperature controlled) Robotic arm Print bed/platform (Temperature controlled) Cartridge/ reservoir

18. Accurate cell distribution and high resolution cell deposition The concept of entrapping green plant cells in a hydrogel is called immobilization. Per say, entrapping green plant cells within bioink prior its deposition for later cultivation could be considered as an advanced cell immobilization technique.

19. Types of Bioprinters : inkjet bioprinting (i.e. droplet-on-demand and continuous inkjet bioprinting), extrusion-based bioprinting (i.e. pneumatic, mechanical, piston or rotating screw-driven extrusion), light-assisted (i.e. digital light processing) and laser-assisted (i.e. laser induced forward motion, laser guided direct writing, two-photon polymerization) biocompatibility, biodegradability and printing resolution (RTM value) - spatial resolution versus time of manufacturing ratio, i . e., the RTM value Two types of Bioprinters inkjet bioprinting consists of the deposition of small droplets of hydrogel and cells microextrusion bioprinting involves cell-laden liquid solution deposition through a nozzle via pneumatic or mechanical force laser-assisted bioprinting process in which a laser point is used to jet cells from a donor surface, landing precisely onto a substrate surface

20. Comparison Between Different type of Bioprinters ?

21. Steps in Bioprinting: Plant cells sources and seeding cell densities concentrated biomass mass (10 to 40g of basil cell plant concentrated biomass per g of bioink. 50% wt per bioink volume of rice plant cells cell densities (2 x 10^6 cell/g of bioink for C. reinhardtii or C. sorokiniana ) single or clustered cells in suspension have been used, micro-extrusion of live cells was only achieved with nozzles of 610µm inner diameter, while smaller nozzle sizes were clogged during the extrusion process.

22. bioproduction of plant cells biomass : growth protocols were inherited from plant tissue culture (PTC) Two types of cell expansion protocols : the growth of unorganized plant cell aggregates : Calli induction is based on the regeneration ability of plant. It is the result of an unorganized proliferation of cells leading to a cell mass from isolated plant cells, tissues, or organs. the growth of single plant cells suspension : protoplast extraction through mechanical or enzymatic treatment. Protoplasts are “naked plant cells” deprived of their wall but still protected by an intact plasma membrane. selecting a healthy plant donor tissue Surface treatment techniques using detergents and disinfectant agents are used to disinfect the explant’s surfaces. sodium or calcium hypochlorite, ethanol, or mercury chloride Sodium hypochlorite solution (bleach) at 5% for 10 to 15 minutes is popularly used for surface disinfection (* disinfection is aggressive treatments several parameters such as detergents concentrations, exposure time has to be optimized) Growth media grow in suspension, cultivation protocols are very close to mammalian cell or microbial suspension cultures. Subculture suspended cell biomass are here cultured in shake flasks with rotating agitation (between 110 and 140rpm). (* providing specific nutritive and physico -chemical environment to the cells to reach optimized growth, temperature 21°C and 28°C and humidity control necessary for cell culture lighting and CO2 diffusion) photoautotrophic (need of a light source and CO2 for photosynthetic energy production) and chemotrophic (growth in the dark on a carbon source) conditions.

23. Parameters of Plant cells cultures: lightning homogeneity, sufficiently transparent to allow for light diffusion, oxygenation hydrogel microporosity should allow for both nutrients, O2 and CO2 access to the cells mass transfer of molecules within the cellularized matrix. Micronutrients soil composition analysis, 17 elements are known to be essential for land plant cell growth - nitrogen (N), potassium (K), calcium (Ca), phosphorus (P), magnesium (Mg), and sulfur (S) with small quantities of minor elements such as iron (Fe), nickel (Ni), chlorine (Cl), manganese (Mn), zinc (Zn), boron (B), copper (Cu), and molybdenum (Mo). Macronutrients carbohydrates, vitamins, and amino acids Growth factors Differentiation factors Bhatia in 2015 Plant MS media, microalgae Tris-Acetate-Phosphate buffer.

24. Bioimaging ADD types of Scanners

25. the main stages of object 3D printing consist of three steps : first step is the creation of a solid in-silico model in CAD software to determine the global geometry of the object. This model is then converted into a standard format for 3D printing. The standard tessellation language format (STL) second step consists of slicing the solid in-silico model. This step will convert the object into a machine file describing the path 3D printer has to follow to deposit or fuse the material. printing parameters (infill density, layer thickness, printing speed) The third step which is the printing process is automated as 3D printer equipment can execute the instruction sequence In case of Plant cells, post-treatment consists in cross-linking of biomaterials forming hydrogels to maintain the printed architectures over cultivation time. shape stability and scalability, Curing and sintered (heat & presure ). Gelation process is commonly based on ionic interaction or fibers re-arrangements to form complex molecules macrostructures

26. Monitoring of cell growth and activity in 3D bioprinted constructs growth, distribution and metabolic activity of cells in bioprinted 3D constructs remains a challenge overcoming O2 limitation is an important issue in tissue engineering and 3D bioprinting cell-biomaterial matrix can lead to steep light gradients limiting photosynthetic growth, a common challenge of dense algal growth in biotechnology. Destructive methods of quantification : Cell biomass, viability and distribution in bioprinted 3D constructs are typically monitored using various types of light microscopy in combination with common live-dead staining protocols and/or through the use of cell autofluorescence or cells tagged with fluorescent indicators or bioluminescent reporters, such as GFP. alginate-based constructs can be dissolved in sodium citrate followed by cell counts with a microscope or flow cytometer non-destructive quantification of cell numbers in bioprinted 3D constructs remains a challenge, Optical coherence tomography (OCT) is a non-invasive, label-free 3D imaging technique that maps structure by measuring the direct backscatter of near-infrared radiation at refractive index mismatches between cells/tissues, extracellular matrix material and the surrounding medium. ultrasound imaging, chlorophyll fluorescence, high-resolution CT scanning

27. Biotechnological concepts using 3D bioprinting of microalgae Microalgae comprise a phenotypically diverse group of photosynthetically active eukaryotes bioconversion of CO2 into extracellular organic products using natural biosorption potential of microalgae for purifying polluted air or waste water immobilization of microalgae resulted in an increased chlorophyll and carotenoid content, increased photosynthetic activity and lipid production (continuous light and 14/10 h light/dark cycles) and temperatures (26 ◦C, 30 ◦C and 37 ◦C), continuous illumination at a photon irradiance (400-700 nm) of 150 μmol photons m-2 s -1

28. Advantages of Bioprinting : Speed of production Food with various geometric shapes Control over nutritional values Texture can be adjusted Reduces environmental pollution Reduces food wastes Advantages of Bioprinting in general : tissue engineering and regenerative medicine (TERM) - treatment of injured or degenerative tissue by fabrication of functional tissue equivalents or disease models, utilizing mammalian cells. reaction compartments, real-time monitoring of the physiological state, growth and metabolic activity of the embedded cells in 3D bioprinted constructs.

29. Advantages of Plant Bioprinting ? reducing waste related to the biomaterial manufacturing process by producing only useful plant components (e.g., secondary xylem or wood) rather than undesirable or unusable plant parts (e.g., leaves, small twigs, roots, or bark). studying subcellular and molecular dynamics within plant cells. Studying Root chemistry - Bioprinted root-based 3D models or biorobots may help clarify the geometrical and mechanical properties of root analogs. Decellularized spinach leaves, for example, can help explain leaf venation Biofiltration or the extraction of heavy metals, nutrients, and industrial pollutants from wastewater is another key application for photoautotrophic microalgae. microalgae are sensitive to a wide range of pollutants, they have been used to develop biosensors for assessing the quality of the aquatic environment.

30. Disadvantages of Bioprinting : Ethical issues Require softwares / trained personnel No control on rheology of food printed Limitations of 3D Bioprinting in Plant Science 3D fabrication of plant parts is still in its infancy due to the lack of appropriate bioink materials difficulty in recreating sophisticated microarchitectures that accurately and safely mimic natural biological activities cell viability long-term functionality accurate process parameters

31. Case Study Paper

32. Commercial BChE extracted from human and animal blood plasma By using Bioprinting of transgenic rice cells, Production of Recombinant Protein rrBChE BChE is used in medicine as preventive and prophylactic drug, it an enzyme found in Human blood plasma acts as bioscavenger rrBChE is tetramerized glycoprotein ~260 KD Objective of this Research: Why didn’t they use Cell suspension culture for synthesis of rrBChE Slower growth rate Formation of cell aggregation Limited mass transfer (low nutrients and gases exchange for the cells) Lower volumetric product accumulation Why didn’t they use animal cells for production of BChE ? Expensive and maintenance is difficult compared to plants Mammalian cells lines easily constitute viral contaminants

33. 0 6 12 14 No. of Days Production phase (Sugar free media) No. of Cells Growth Recovery Phase NB+S NB-S NB+S

34. They have checked for cross linking efficiency/ curing using UV (long wavelength) for an hydrogel 12 % (PEGTA) Which maintains structural integrity

35. 0 6 12 14 No. of Days Production phase (Sugar free media) TTC assay Growth Recovery Phase Dark Red Light Red Dark Red Total Cell viability Test (Triphenyl tetrazolium chloride) Solid pieces of hydrogel 0.2g , washed with ddh20 0.8 ml ttc , incubate dark 12-24 hours (RT, without shaking) Remaining TTC was discarded Washed with ddh2o And add 1.5 ml of 95% reagent ethanol to extract triphenyl formazan into supernantant @ 60c heat block Dark red colour indicates cells are viable Supernanant spectrophotometric analysis 485 nm absorbance normalized to g FW of the cells

36. LIVE/DEAD Ratio Fluorescent Measurement and Confocal Microscopy for Cell Viability The green channel measures viable cell signal with SYTO 9 stain (excitation/emission: 470/540 nm) while the red channel measures non-viable cell signal with propidium iodide (excitation/emission: 470/620 nm). stained cells were imaged using a Zeiss LSM 700 confocal microscope. Live/dead SYTO9 stain (attach to live active nuclei)/ propodium iodide (attached to cell wall and dead nuclei) Curing without Curing Curing with Curing

37. Bioprinting of plant cells for production of a biodefense agent: Transgenic rice cells were immobilized using this bioink ( Oryza sativa ). This is the first report of recombinant plant cells being immobilized for the continuous synthesis of high-value heterologous proteins. The preparation of the suspension culture of rice cells Encapsulation was performed using a transgenic O. sativa rice cell line expressing recombinant rice butyrylcholinesterase ( rrBChE ) Rice cells were cultured and grown in a semi-solid “NB+S” selection medium including N6 macronutrients, B5 micronutrients, and vitamins, 30 g l−1 sucrose, 1.8 g l−l Gelzan ™, 300 mg l −l caseinhydrolysate , 250 mg l−l L-glutamine, 250 mg l −l L-proline, 2 mg l−l 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.02 mg l−l Kinetin, with 50 mg l−l geneticin as the selection antibiotic.

38. The cells were subcultured into liquid sterile NB+S media start of the experiments by pressing and sieving the calli through a sterile, stainlesssteel , 280 m mesh sieve to achieve consistent cell aggregates, incubated in the dark at 28°C, 140 rpm The bioink used to immobilize the rice cells contained 12% (w/v) 4-arm polyethylene glycol tetraacrylate MW 20,000 (PEGTA), with 0.1% (w/v) LAP as the photo initiator Experiments were carried out in a biosafety cabinet with the bioink extruded manually using a 1 mL sterile Luer -lock syringe furnished with an ~840 μm inner diameter tapered tip The results suggested that this bioink (polyethylene glycol-based hydrogel) successfully immobilized transgenic rice cells (Oryza sativa) producing recombinant butyrylcholinesterase, which acts as a prophylactic or therapeutic against cocaine toxicity, neurodegenerative disorders such as Alzheimer’s disease and organophosphate nerve poisoning.

39. PEG entrapping /immobilizing stable transgenic rice lines grown for 14 days.hhhhh Growth phase 0-6 days Production phase 6-14 days (Sugar free media growth) Growth/recovery phase (12-14) days rrBchE secreted in the media, thus easy for extraction, thus plants used for extraction of high value heterologous protein.

40. rrBchE How it works ? Rice alpha amylase (RAmylas3D) promoter- rrBchE gene expresses undersugar starvation condition. And has signal peptide that tags proteins secreted by rice cell lines. Media Rice cells were cultured and maintained according to previously described procedures on semi-solid “NB+S” selection medium containing N6 macronutrients, B5 micronutrients and vitamins, 30   g/L sucrose, 1.8   g/L Gelzan ™, 300   mg/L casein hydrolysate, 250   mg/L L-glutamine, 250   mg/L L-proline, 2   mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.02   mg/L kinetin, and 50   mg/L geneticin as the selection antibiotic. WORK FLOW OF RICE CELL LINES USED FOR PRODUCTIONOF rrBchE : Rice cell lines grown in NB+S media Callus pressed against 280 um stainless stain sieve to get small and consistent cell aggregates Put back to suspension media take 500 ml of susepension in 1 L shake flasks (working volume) Incubate @ 28 c , 140 rpm in dark condition.

41. Bioink preparation : PEGTA and LAP ( Photoiniator ) , extrusion 840 um diameter syringe. 0-6 (growth phase)+ sugar media, 6-12 th day – production phase (Sugar starvation medium NB-S medium), 12-14 days (Revival phase + sugar medium)

42. Crude cell extract The cells embedded in the hydrogel were more difficult to homogenize, and it took ∼ 180 seconds to crush both the hydrogel and the cells. The homogenized samples were centrifuged again for a minimum of 10 minutes, and the supernatant containing rrBChE was transferred to a new tube for further tests. Tubes of crude extract and of media were stored at 4°C or -20°C for short-term use or at -80°C for long-term storage.

43. SDS-PAGE (Protein qualitative analysis) and western blot analysis [presence and confirmation of monomer size ~ 65 KDa ](Antibody with probe mouse anti- BChE HRP ) Amylase (~45kDa) and rrBchE are coregulated by same RAmyl3Dpromoter.

44. Case study 2

45. studying cellular reprogramming and cell cycle reentry toward tissue regeneration. Capturing cell-to-cell signals in a three-dimensional (3D) environment is key to studying cellular functions. They have studied 3D bioprinting plant cells cell viability, cell division, and cell identity by analysing large image datasets by high-throughput image analysis pipeline. They also showed how cell cycle reentry of bioprinted cells happening and how its coinciding induction of core cell cycle genes and regeneration related genes leading to formation of microcallus . For tissue regeneration there must be coordination between cellular reprogramming ie dediffr . Of cells into pluripotent state and cell cycle progression via cell-cell communication is affected by physical and molecular interaction between cells and their microenvironment   conventional tissue culturing methods do not entirely mimic the 3D microenvironment observed under natural conditions hence, it is difficult to study cellular functions , so 3d constructs better simulates natural in planta conditions Plants inherently show extreme regenerative capacities, The regenerative capacities of individually isolated plant cells are greatly affected by cell type, tissue type, genotype, and the specific 3D microenvironment

46. Materials: thaliana seeds were surface-sterilized using 50% bleach and 0.05% Tween 20 for 5 min, followed by a 2-min incubation in 70% ethanol for 2 min. Seeds were rinsed at least seven times with sterile deionized water. Following the last rinse, the seeds were stratified at 4°C for 2 days and plated on 1× Murashige and Skoog agar supplemented with sucrose (1% sucrose total) on top of Nitex mesh (Genesee). All plants were grown in a vertical position at 22°C under long-day conditions (16-hour light/8-hour dark cycle). Soybean [G. max (L.) Merr . cv Thorne] seeds were surface-sterilized using 70% ethanol for 2 min, followed by a 10-min incubation in 50% bleach and 0.05% Tween 20. Seeds were rinsed 10 times in sterile deionized water. Following the last rinse, the seeds were placed in a sterile petri dish, covered with sterile deionized water, and imbibed at 27°C under long-day conditions (16-hour light/8-hour dark cycle) for 24 hours.

47. Isolation of protoplasts To avoid contamination, all steps were performed in a laminar flow hood. To prepare the enzyme solution, 0.45 g of cellulase (EMD Millipore) and 0.03 g of pectolyase (Sigma-Aldrich) were dissolved in a 30-ml solution containing 5.465 g of mannitol, 0.05 g of 0.01% bovine serum albumin, 500  l of 0.2 M magnesium chloride, 500  l of 0.2 M calcium chloride, 500  l of 1 M MES, 500  l of 1 M potassium chloride, 50 ml of deionized water, and tris-HCl (pH 5.5). The enzyme solution was sterilized using a 0.20-  m syringe filter. For each sample, 7 ml of fresh enzyme solution was pipetted into 35-mmdiameter petri dishes. We avoided creating bubbles when pipetting the enzyme solution to avoid cell lysis at later stages in the protocol. A 70-  m cell strainer was placed in each 35-mm-diameter petri dish. Approximately 1 to 2 mm of the root tip were cut to isolate the meristematic region of the root and put into the strainer in enzyme solution. The samples were incubated for 2 hours at 85 rpm at room temperature with supplemental stirring every 30 min. Next, all of the cells and enzyme solution were transferred to a 15-ml conical tube and centrifuged for 6 min at 200g. The supernatant was removed, and the pellet was resuspended with 100  l of PIM or protoplast callus induction medium (PCIM) for the shoot-derived cells (47, 48). PIM (A. thaliana) contains a cocktail of growth hormones, sugar, mannitol to ensure a proper osmolarity, folic acid (18, 19), and PSK (17, 49) (Table 1). The resuspended solution was transferred to a 70-  m filter placed on top of a 50-ml conical tube. Another 100   l of PIM was filtered through to collect any leftover cells stuck on the 70-  m filter. All the filtered liquid was transferred to a 40-  m filter placed on top of a 50-ml conical tube. The subsequent filtered liquid contains the protoplasts used for bioprinting. The volume of cells in each solution was estimated using a hemocytometer.

48. Arabidopsis root–derived protoplasts, i.e., plant cells without cell wall, Protoplasts are an alternative system to callus and suspension cultures that exhibit individual cellular behavior.   They have 3 d cultured prothoplast of arabhidopsis observed for 7 days, they could found that endodermal markers increses during that period, indicating identity of isolated cells changes over longer periods of time. They have taken cells from two diff tissue arabhidopsisgain insights into whether different tissue types would be more prone to cellular reprogramming, we isolated protoplasts from both meristematic and differentiated root tissue and performed comparative analyses . To process and analyze these large image datasets generated from our bioprinting pipeline, we developed a semiautomated and high-throughput image analysis pipeline that can quantify cells in confocal z-stack images (Screenshot of the graphical user interface (GUI) of our analysis pipeline for high-throughput quantification of cells in confocal z-stack images .)

49. Tissue-specific bioinks require the balancing of auxin and cytokinin, which are critical for long-term cell viability and proliferation added phytosulfokine (PSK) and folic acid, two compounds that stimulate cell proliferation, After bioprinting cells from two diff tissues 62.2 ± 8.2% and 51.6 ± 8.3% viable meristematic and differentiated protoplasts immediately (day 0). After 5 days, generally 27.7 ± 7.6% and 29.5  ±  3.5% of the meristematic and differentiated isolated cells were still viable

50. When stained with propidium iodide, a contrast staining for cell death, similar cell viability percentages were obtained. ell viability is reducing day to day, my be the post bioprinting phase conditions causing cells to die.

51. Applicability of 3D bioprinting toward plant cell regeneration   Wheather the cell division is happening in bioprinted cells or not ? Bioprinting of isolated cells into desired shapes helps to study cellular regeneration by callus formation from root explants initiates with divisions of pericycle cells, rather than dedifferentiation and cell cycle reentry of root cells cell cycle reentry of protoplasts derived from Arabidopsis roots has not yet been achieved They observed bioprinted Arabidopsis root cells undergo their first cell division as early as 3 days To map the cells’ potential to divide, we used the cell cycle marker pCYCB1;1:CYCB1;1-GFP, which is expressed at the transition from the G2 gap phase into mitosis (M phase), After a 7- to 10-day period, ~12% of the bioprinted constructs developed a microcallus , here defined as four cells or more, they also collected RNA from isolated differentiated root cells on days 0, 1, and 3 after bioprinting. revealed 9071 differentially expressed genes (DEGs) identified seven clusters of coexpressed genes, From left to right: High-resolution confocal images of isolated protoplasts, isolated protoplasts undergoing cell division, and microcalli from Arabidopsis roots, shoots, or soybean shoot meristematic regions Anisotropic elongate

52. Number of formed microcalli for Arabidopsis roots and soybean shoot meristematic region. Each dot represents a bioprinted construct Percentage of green fluorescent protein (GFP)–expressing pCYCB1;1:CYCB1;1-GFP meristematic and differentiated root cells on days 0, 1, 3, 5, and 7.

53. Heatmap of the transcriptional changes for significantly differentially expressed core cell cycle (left) and regeneration (right) genes 0, 1, and 3 days upon bioprinting of differentiated root cells.

54. Identity maintenance of 3D bioprinting root cells The root contains multiple cell types, and thus, the bioprinted root cell population is highly heterogeneous. To shed light on the identity of the isolated cells that remain competent. ground tissue identity and tracked it using cell type– specific marker lines. Two cell types endodermis (pEN7:GFP (pENDODERMIS7:GFP),) and cortex endodermis initial (CEI) cells ( pSCR:SCR-mCherry )

55. Biotechnological concepts using 3D bioprinting of plant cells Plants produce secondary metabolites which are valuable agents for the pharmaceutical, food, agrochemical and cosmetics industries. Natural secondary metabolites useful for medical applications encompass 1) alkaloids that are used in a broad range of applications such as pain killers or for treatment of cardiac diseases, hypertension, malaria, Alzheimer’s disease, gout or glaucoma, 2) di/tri-terpenes that are used as anti- tumour or eco-friendly antimicrobial and insecticidal agents, and 3) polyphenols with antioxidative, immunomodulatory, anti-inflammatory and anticancerogenic activities

56. traditional extraction of the secondary metabolites from agricultural or wild plants, which is connected with strong fluctuations in quality and quantity due to varying biotic and abiotic environmental factors production of the anti-tumor agent paclitaxel by Taxus sp. secondary metabolites are usually accumulated within the plant cells at comparably low concentrations. Strategies to increase the resulting low product yield of plant cell cultures are 1) elicitation, i.e., the introduction of chemical or physical stress to induce synthesis and/ or secretion of secondary metabolites as a defence reaction, 2) in situ product removal, in order to avoid feedback inhibition of product synthesis and product degradation, 3) immobilization by encapsulation of plant cells – mostly in biomaterials – to protect them from shear, to achieve a high cell density and to allow continuous product removal, and 4) metabolic engineering using DNA transformation techniques

57. So in above the 3 rd point is 3D bioprinting for secondary metabolite production is used. The structured immobilisation of plant cells in 3D constructs with a defined porous architecture enables. continuous removal of the products can be easily realized without loss of cells and two-stage processes, with a growth, phase followed by a production phase, can be easily carried out by changing from a growth to a production medium environment. The bioink was produced by mixing callus-derived cell aggregates of basil ( Ocimum basilicum ) into a biomaterial ink consisting of 3 wt % alginate, 3 wt % methylcellulose and 1 wt % agarose (AMA), and processed into 3D constructs with high shape fidelity by using extrusion-based 3D bioprinting. After alginate crosslinking in CaCl2 solution, the 3D structures were cultivated for 20 days. The embedded basil cells showed a high viability and proliferation, indicated by fluorescence staining and sugar consumption of the heterotrophic cell culture.

58. Future Perspectives of 3D Bioprinting ? 4D printing Reaction Compartments - structured immobilization matrices could be easily integrated into bioreactors (fixed-bed reactors) Future prospects of Green bioprinting : extraction of high-value compounds in the biopharmaceutical field process of in vitro redifferentiation is generally associated with an improved synthesis of secondary metabolites in vitro land-free production strategies green bioprinting might help reduce the competition between arable and forested areas 3D bioprinting product subject to change in shape, colour and flavour over time 4D biopriting with induced by internal and external stimulus (pH, h20, heat) Food printing of astronauts for long duration missions ISS, Alpeh farms 3D printed meat and Russia space organization ROSCOSMOS

59. Bioethics of Bioprinting : Concern over toxicity of biomaterial Food of animal origin – fullfillment of moral validity

60. References : LANDERNEAU, S., LEMARIÉ, L., MARQUETTE, C. AND PETIOT, E. , 2022, Green 3D bioprinting of plant cells: A new scope for 3D bioprinting. Int. J. Bioprinting ,  27: 1-2 MEHROTRA, S., KUMAR, S., SRIVASTAVA, V., MISHRA, T. AND MISHRA, B.N., 2020, 3D bioprinting in plant science: An interdisciplinary approach.  Trends Plant Sci. ,  25 (1). VARMA, A., GEMEDA, H.B., MCNULTY, M.J., MCDONALD, K.A., NANDI, S. AND KNIPE, J.M., 2021, Bioprinting transgenic plant cells for production of a recombinant biodefense agent.  bioRxiv , 02: 21-27

61. GHOSH, S. AND YI, H.G., 2022, A Review on Bioinks and Their Application in Plant Bioprinting.  Int. J. Bioprinting ,  8 (4). KRUJATZ, F., DANI, S., WINDISCH, J., EMMERMACHER, J., HAHN, F., MOSSHAMMER, M., MURTHY, S., STEINGRÖWER, J., WALTHER, T., KÜHL, M. AND GELINSKY, M ., 2022, Think outside the box: 3D bioprinting concepts for biotechnological applications–recent developments and future perspectives.  Biotechnol . Adv . ,  58 : 1202-1209 VAN DEN BROECK, L., SCHWARTZ, M.F., KRISHNAMOORTHY, S., TAHIR, M.A., SPURNEY, R.J., MADISON, I., MELVIN, C., GOBBLE, M., NGUYEN, T., PETERS, R. AND HUNT, A ., 2022, Establishing a reproducible approach to study cellular functions of plant cells with 3D bioprinting.  Sci. Adv . ,  8 :41 . References :