There are many demands for the the bioprinter prototype, based on the scientific literature and research activities of the predecessors and actual project partners, from the medical and biological approach, for the viability and functionality of the cells, during and after the deposition of biomaterials.
The designated medium in which islet cells will be provided will be a modified RPMI 1640 medium (standard cell culture medium). Modifications will be done to reduce apoptosis and de-differentiation into earlier maturation stages. E.g. for supplements (Daoud, J., Rosenberg, L. and Tabrizian, M. (2010) ‘Pancreatic Islet Culture and Preservation Strategies: Advances, Challenges, and Future Outlook’, Cell Transplantation, 19, pp. 1523–1535. doi: 10.3727/096368910X515872):

  • Glucose, 5.6 mM (optimal concentration for human islets)
  • Ascorbic acid, <200 μM (role in glucose sensing, restores Vit. E)
  • Zinc, 16.6 μM (Proinsulin requires zinc for storage)
  • IGF-I, IGF-II (Cell differentiation, cell growth, tissue repair)
  • L-Glutamin, 6.8 mM (preserves ability of islets to regulate o-glycosylation)
In summary, none of the supplements that we propose for usage in vitro cell culture of human islet cells will change the viscosity, the culture conditions (37°C, 5% CO2) or the requirements for a bioprinter if compared to standard medium. The medium will be used for in vitro cell culture prior printing and to perfuse the partially printed construct intra-printing.
For the printing process itself we propose different strategies for scaffolding. Islet cells will be either embedded in scaffold materials (hydrogels) or seeded on printed scaffolds or combination of both. In the last step an addition as well as in vivo ingrowth of epithelial cells by perfusion will be promoted by ECM-functionalized surfaces on the designated micro channels within the scaffold and can be supported with retarded release of scaffold-bound growth factors (e.g. vascular endothelial growth factor) for sprouting of new vessels. Our systematic review of the literature (Salg, G et al.The Emerging Field of Tissue Engineering: An Evidence Map and Systematic Review of Scaffold Materials and Scaffolding Techniques for Pancreatic Islet Cells. To be published. Confidential) that have been published in the last decade revealed that it is crucial for cell viability and function (especially in human islet cells) to mimic the extracellular matrix for successful organ creation approaches. In native tissues ECM contributes to viability and function of cells by (I) providing structural properties, (II) ensuring mechanical stability, (III) regulating cellular activities, (IV) storing and releasing growth factors and by (V) providing a degradable environment that can be remodeled on demand (Chan BP, Leong KW. (2008). Scaffolding in tissue engineering: general approaches and tissue-specific considerations. Eur Spine J. 17 Suppl 4:467–79). To replicate each of these ECM functions, biological and synthetic materials as pre-made porous scaffolds for cell seeding, decellularized ECM-scaffolds, scaffold cell sheets and cell encapsulating hydrogels are currently used. The application of scaffolds range from the generation of insulin-producing cells from stem cells and progenitor cells in scaffold based 3D-culture systems to building artificially-created support systems that serve as a logistic template, prevent anoikis, protect from inflammatory and immunological host reactions and improve long-term viability. According to positive results in past in vitro and in vivo studies 18% used alginate, 14% used PEG, 5% used PLGA and 3% used PCL. All these materials showed promising results to conduct further research. Please note, that bioprinting was only used twice in the publications that met the inclusion criteria (representing <1% of the articles) and therefore frequencies are only partially consequential.
In consideration with the literature search we propose a multi-modal approach in 3D-PIVOT. Proof of function, in vitro and in vivo, validation and streamlining of the process can only be investigated once the bioprinting hardware will be provided. The current research plan after delivery of the hardware includes the testing of following materials for usage in scaffolds.

  • Alginate
    (Marchioli G et al. (2015). Fabrication of three-dimensional bioplotted hydrogel scaffolds for islets of Langerhans transplantation. Biofabrication.7:025009) (Kojima N, Takeuchi S, Sakai Y. (2014). Engineering of Pseudoislets: Effect on Insulin Secretion Activity by Cell Number, Cell Population, and Microchannel Networks. Transplantation Proceedings. 46:4. 1161-1165. doi.org/10.1016/j.transproceed.2013.11.147.)
  • PEG
    (Phelps, EA et al. (2015). Engineered VEGF-releasing PEG-MAL hydrogel for pancreatic islet vascularization. Drug delivery and translational research. 5:2. 125-36.) (Skardal, A et al. (2016). Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink. Journal of visualized experiments : JoVE, (110), e53606. doi:10.3791/53606) (Jamal, M et al. (2013), Bio‐Origami Hydrogel Scaffolds Composed of Photocrosslinked PEG Bilayers. Advanced Healthcare Materials. 2: 1142-1150. doi:10.1002/adhm.201200458)
  • Commercially available bioink hydrogel for positive control
    (e.g. CellInk A-RGD, CellInk LAMININK 521 – both https://cellink.com/bioink; Alginate-RGD acellular bioink, Sigma Aldrich – 901950)
  • PCL
    (Marchioli, G et al. (2016). Hybrid Polycaprolactone/Alginate Scaffolds Functionalized with VEGF to Promote de Novo Vessel Formation for the Transplantation of Islets of Langerhans. Adv. Healthcare Mater., 5: 1606-1616. doi:10.1002/adhm.201600058) (Mohammad FA et al. (2018). PCL/PVA nanofibrous scaffold improve insulin-producing cells generation from human induced pluripotent stem cells. Gene. 671:50-57. doi.org/10.1016/j.gene.2018.05.115)
  • PLGA
    (Jamal T. et al. (2011). Long-term in vitro human pancreatic islet culture using three-dimensional microfabricated scaffolds. Biomaterials.32: 6. 1536-1542.doi.org/10.1016/j.biomaterials.2010.10.036) (Mironov AV et al. (2017). 3D printing of PLGA scaffolds for tissue engineering. J Biomed Mater Res Part A. 105A:104–109)
  • Sacrificial scaffold material to be solved by water
All of the materials mentioned above are subject to change and can be modified and supplemented for better cell attachment properties, hydrophilicity and printability (see cited publications). We see the necessity for our approach to provide protection for the cells from the immune system. In current therapies for islet transplantations into the liver veins following the Edmonton protocol a large part of the islet cells will go into apoptosis due to IBMIR (instant blood-mediated inflammatory reaction). In the long term auto-antibodies and delayed immunoreactions will further compromise the amount of viable islets. We have the reasonable assumption that the use of a single scaffold material within the bioartificial organ will not be sufficient. The combination of two or more materials should give mechanical stability to the organ, protect cells from the immune system and host them in a way that mimics natural pancreatic cell surroundings. The designated implantation site for the bioartificial organ, created within the 3D-PIVOT project will be different from the most usual implantation sites described in the literature and used for preliminary trials. Over 30% of the published in vivo experiments chose a subcutaneous implantation. Others used the peritoneal cavity, omental pouch or subrenal capsula for implantation of their INS+ device. As hypoxia and a lack of nutrient supply is the most relevant cause for apoptosis on the long term, we propose the implantation of our bioartificial device in close proximity to vascular structures with easy surgical access. It will then benefit from the connection to natural perfusion systems instead of hypoxic diffusion in fatty tissue and still will not require extensive and possibly harmful surgery. Besides the eligibility of the printer to process the above materials, there are requirements for the printing process as well. To maintain viability and ensure sufficient oxygen and nutrient supply islet cells should be in close proximity to a supplying structure (vessel, microchannel). Literature search revealed 150-200μm as a maximum upper distance limit for e.g. cells encapsulated in hydrogel (Park, J. , Kalinin, Y. V., Kadam, S. , Randall, C. L. and Gracias, D. H. (2013), Microfabricated Bioartificial Pancreas. Artificial Organs, 37: 1059-1067. doi:10.1111/aor.12131). Considering the size range of a human islet (50-250 μm) these parameters represent the printing precision that will be necessary for the bioprinter. The fact, that precise scaffolding is not only influenced by the printing hardware but also by the scaffold material (viscosity, gelation time and crosslinking technique) is understood.

Bioprinter concept/design (LTHD Corporation):

The main components that are developed, designed and manufactured are shortly presented in the following, due to the limitation of the text only interface in myeureka.io:

Blocul electronic de acționare al motoarelor de acționare a pistoanelor seringilor de injecție, respectiv de termo-condiționare a temperaturii capului de depunere al materialelor termoplastice se află în gestiunea modulelor electronice, care, de asemenea trebuie interconectate. În figurile de mai jos sunt prezentate aceste plăci electronice, atât de la nivelul elementelor de acționare cât și de la nivelul volumului de lucru (incubatorului).

Pentru incubator, echipamentul electronic, dezvoltat în jurul microcontroler-ului STM32F030CC, poate gestiona (monitorizare și control) următorii parametrii (prin interfațarea cu placa de bază prin port-ul RS485):

  • tensiuni de lucru pentru traductori;
  • interfațare cu senzorii de umiditate și concentrații de CO2 și O2;
  • temperatură incintă (acționare la nivel modul Peltier), umiditate, nivel de CO2 și O2 față de valorile de referință;
  • turații ventilatoare de vehiculare aer în incinta imprimantei;
  • nivel de umiditate, prin acționarea combinată a sistemului de umectare și dezumidificare (bazat pe același sistem Peltier, pentru condensarea umidității excesive);
  • controlul pompelor peristaltice pentru dozarea O2 și CO2;
  • controlul temperaturii incubatorului, dotat cu element de încălzire de tip film;