Open Insulin Literature Review

Literature review in progress

Key words terms (KWT)

1. Open Source Insulin
2. Open Insulin Bioreactor
3. Insulin Bioreactor -transplant*

Strategies

1. Searched Google Scholar using KWT1, KWT2, and KWT3

Open Insulin is an initiative to produce affordable, open-source insulin. It involves community engagement, sharing open-source biotech methods, and aims to lower production costs and improve accessibility for diabetics globally. The project focuses on sustainability and collaborative innovation to address the insulin access crisis.

1. Utilization of solid Open Source Bioreactors to generate a three-dimensional suspension culture system of human pancreatic progenitor cells from human iPSCs. Next, differentiated β-cells will produce insulin in response to increased glucose in vitro. The produced insulin will then require purification.

Open Insulin is crucial because it addresses the high cost and limited accessibility of insulin. By developing affordable, open-source production methods, it ensures that life-saving insulin is available to underserved and marginalized communities. The project's transparency fosters trust and continuous improvement, while community engagement empowers those affected by diabetes. Additionally, Open Insulin promotes biotechnological innovation, potentially benefiting broader medical and biotechnological fields.

The Open Insulin project focuses on refining insulin production using genetically engineered microorganisms, producing small batches in labs, engaging a global network, sharing open-source protocols, securing funding, and addressing regulatory challenges. Similar initiatives include Civica Rx, which aims to produce affordable generic insulin; iGEM teams specifically working on synthetic biology solutions for insulin production; Fair Access Medicines, developing low-cost insulin; BioBricks Foundation, supporting open-source insulin production projects; and Open Bioeconomy Lab, creating open-source tools for affordable insulin production. These organizations all aim to improve insulin affordability and accessibility through transparency and innovation.

The relevant stakeholders of the Open Insulin project include diabetic patients, healthcare providers, researchers, non-profit organizations, pharmaceutical companies, regulatory agencies, funding bodies, patient advocacy groups, academic institutions, and policymakers. These groups collaborate to improve insulin affordability and accessibility through open-source production, financial support, scientific advancements, and favorable healthcare policies.

The applicability and context of the Open Insulin project lie in its potential to provide affordable and accessible insulin, addressing the global issue of high insulin costs and limited access. It is particularly relevant in regions with significant diabetic populations and limited healthcare resources, such as low- and middle-income countries. The project can also benefit underserved communities in high-income countries where insulin prices are prohibitively high. The open-source approach allows local production and adaptation, making it feasible for diverse settings, including community labs, academic institutions, and non-profit organizations globally.

CGM= Continuous glucose monitoring

ECM= Extracellular matrix

• heterogeneous population of neonatal porcine pancreatic cells obtained from SMRI (pancreas from 1-3 year old piglets) (Chawla, et. al)
• Commercial and in‐house human‐induced pluripotent stem cell (hiPSC)‐derived pancreatic cells expressing NK6 homeobox 1 (NKX6‐1)+ /pancreatic and duodenal homeobox 1 (PDX‐1)+ were seeded onto scaffolds and evaluated.hiPSC‐derived pancreatic cells were generated following an established stepwise protocol. Briefly, hiPSCs (F3.5.2; Chang et al., 2015) were cultured in mTesr‐1 (STEMCELL Technologies) in matrigel‐coated six‐well plates to reach 80% confluence. When confluent, cells were incubated with Accutase (STEMCELL Technologies) for 5 min to detach and pipetted to form single cells. Single cells were centrifuged at 300g for 5 min; the supernatant was removed, and cells were loaded into custom‐made polydimethylsiloxane microarrays prepared by soft lithography (Tomov, Olmsted, & Paluh, 2015) with 500‐µm wells to form embryoid bodies (EB). After 4 days, EBs were transferred to matrigel‐coated Petri dishes and differentiation initiated. Approximately 30 EBs were plated in each well of a six‐well plate. From Days 1 to 12 of differentiation, Kroon's protocol (Kroon et al., 2008) was followed to generate endocrine precursors. On Day 12, Millman's protocol (Millman et al., 2016) was initiated to further differentiate the cells to mature β cells. Primary pancreatic cells were purchased from Celprogen (Cat# 35002‐04; Torrance, CA) and cultured on precoated flasks (Cat# E35002‐04; Celprogen) according to manufacturer's protocol. (Amini, et. al)
• the human iPSC 253G1 strain was purchased from RIKEN (Tsukuba, Japan) and maintained in Primate ES Cell Medium (ReproCELL, Yokohama, Japan) supplemented with 5 ng/ml basic fibroblast growth factor (ReproCELL) on mitomycin C-treated mouse embryonic fibroblasts (ReproCELL). Cells were passaged as small clumps every 3–4 days using dissociation solution for human ESCs/iPSCs (ReproCELL). (Mihara, et. al)
• INS-1 cells (C0018007) purchased from Addexbio Technologies (California, USA) Hank's balanced salts solution (HBSS, H1387), HEPES (H3375), sodium pyruvate (S8636) and β-mercaptoethanol (M7522) were obtained from Sigma-Aldrich (ON, Canada). Phosphate buffered saline (PBS, BP 665-1) and sodium bicarbonate (MSX03201) were acquired from Fisher Scientific (Ottawa, ON, Canada). RPMI-1640 with l-glutamine (31800-022), penicillin-streptomycin (15140-122) and trypsin-EDTA (25200-056) were purchased from Invitrogen (Burlington, ON, Canada).(Sharp)

• 125 mL suspension bioreactors (spinner flasks, Corning Glass, New York) containing 100 mL of medium. These glass vessels each had two sidearms, which were used for charging the vessel with medium, for inoculation of cells, and for taking samples. Suspension bioreactors were placed on slow speed magnetic stir plates (Thermolyne Cellgro, Barnstead International, Dubuque, IA) at 100 rpm in an incubator operating at 37 °C with 5% CO2. The inner surfaces of all suspension bioreactors and the outer surface of the magnetic stir bars were siliconized with 10% Sigmacote (Sigma-Aldrich, cat. no. SL-2) in hexane (VWR International, cat. no. B90210). (Chawl, et al.)
• wicking matrix bioreactor, fabricated by Sepragen Corporation for scale‐up biomanufacturing of hiPSC‐ derived pancreatic cells. The bioreactor consists of a porous amine‐ modified (A) cellulose scaffold with 20–50‐µm‐wide fibers in a sterile chamber with independent air and media inlets and a waste removal outlet(Amini, et. al)
• wicking matrix cellulose scaffold - Amine‐modified - On the basis of ELISA results, single cells seeded on scaffolds secreted higher amounts of insulin than aggregates cultured on scaffolds, which in turn secreted more insulin than cells cultured on tissue culture dishes, with the highest amount produced by single cells on uncoated A scaffolds.- ECM production is a necessary step for support of multiple cell activities on a scaffold including survival and migration, and in some cases, ECM components can integrate into the scaffold structure to enhance cell attachment, differentiation, and proliferation for tissue engineering(Amini, et. al)

• ultrasensitive insulin ELISA kit (ALPCO 80‐INSHUU‐E01.1)(Amini, et. al)
• Validate differentiation by immunocytochemistry for relevant intracellular pancreatic biomarkers, including PDX‐1—a posterior foregut biomarker, NGN3—transiently expressed at endocrine committed cells, NeuroD1—expressed downstream of NGN3 in pancreatic progenitors, and NKX6‐1—pancreatic endocrine specification of cells (Haller et al., 2019; Hogrebe, Augsornworawat, Maxwell, Valezco‐Cruz, & Millman, 2020; Figure 2a).(Amini, et. al)
• MTT assay(Amini, et. al)
• YSI biochemical analyzer(Amini, et. al)

• Insulin Patents
• Solid OS bioreactors
• Hardware features - sensors
• equipment to purify
• how to test and tools required to test

Braune, K., Lal, R. A., Petruželková, L., Scheiner, G., Winterdijk, P., Schmidt, S., ... & Hussain, S. (2022). Open-source automated insulin delivery: international consensus statement and practical guidance for health-care professionals. The Lancet Diabetes & Endocrinology, 10(1), 58-74.

• Summarizes current evidence and describes relevant technologies for AID
• Highlights global healthcare consensus and provides clinical guidance on adopting open-source systems in medical settings.
• Offers key recommendations for stakeholders in diabetes technology but mostly focuses on Open Source automated insulin delivery algorithms rather than Open Source Insulin Production

Gallegos, J. E., Boyer, C., Pauwels, E., Kaplan, W. A., & Peccoud, J. (2018). The Open Insulin Project: A case study for ‘biohacked’ medicines. Trends in biotechnology, 36(12), 1211-1218.

• The Open Insulin Project is developing a protocol for insulin production to bypass intellectual property restrictions.

Amini, N., Paluh, J. L., Xie, Y., Saxena, V., & Sharfstein, S. T. (2020). Insulin production from hiPSC‐derived pancreatic cells in a novel wicking matrix bioreactor. Biotechnology and Bioengineering, 117(7), 2247-2261.

• Static, wicking matrix bioreactor, provides a thin film of medium dripped onto cells on the scaffold, offers advantages for 3D culture such as improved oxygen transfer without the detrimental properties of dynamic systems.
• This study found the optimal conditions to produce insulin from hiPSC-derived pancreatic cells however insulin production seemed to decline by day 5 and 12, consistent with loss of cells from the scaffolds. Insulin release increased significantly with increasing glucose concentration
• Insulin secretion by scaffold‐attached hiPSC‐derived NKX6‐1+ /PDX‐1+ pancreatic cells increases during bioreactor culture, this showed the greatest potential for cell expansion and insulin production
• Differentiating pancreatic cells expanded 10‐fold in bioreactor while maintaining steady metabolic activity over 13 days
• Tested multiple surface modification conditions for cell attachment and expansion in a multiwell test platform and small‐scale bioreactor

Mihara, Y., Matsuura, K., Sakamoto, Y., Okano, T., Kokudo, N., & Shimizu, T. (2017). Production of pancreatic progenitor cells from human induced pluripotent stem cells using a three-dimensional suspension bioreactor system. Journal of tissue engineering and regenerative medicine, 11(11), 3193–3201. https://doi.org/10.1002/term.2228

• Differentiated β-cells secreted insulin in response to increased glucose in vitro.
• Three-dimensional suspension culture system can generate human pancreatic progenitor cells from human iPSCs
• Further optimization of culture conditions should provide sufficient functional islet cells for transplantation therapy
• Examined the ability of differentiated β-cells to secrete insulin in response to increased glucose concentrations in vitro. Total levels of human-specific C-peptide were significantly increased in cells cultured in high-glucose medium compared with low-glucose medium suggesting that iPSC-derived β-cells could secrete insulin in response to elevated glucose concentrations

Magrofuoco, E., Elvassore, N., & Doyle III, F. J. (2012). Theoretical analysis of insulin‐dependent glucose uptake heterogeneity in 3D bioreactor cell culture. Biotechnology progress, 28(3), 833-845.

• This study describes a mathematical model to assist the experiment design of the relationship between biochemical stimuli and cell response within a 3D cell culture in scaffold with heterogeneous porosity, specifically the effect of insulin on the local glucose metabolism as a function of 3D pore size distribution.
• Found that the flow rate is the most important operative variable, and that an optimum value ensures a fast and detectable cell response to produce insulin

Chawla, M., Bodnar, C. A., Sen, A., Kallos, M. S., & Behie, L. A. (2006). Production of islet‐like structures from neonatal porcine pancreatic tissue in suspension bioreactors. Biotechnology progress, 22(2), 561-567.

• Found that new serum-free medium developed in their laboratory was capable of supporting endocrine cell expansion, including insulin positive cells that were glucose responsive
• 125 mL suspension bioreactors operated at 100 rpm in an incubator maintained at 37 °C and 5% CO2
• PPRF-p1 could support higher cell densities during the 9-day
• bFGF has also been shown to act in a paracrine manner with human pancreatic cells, stimulating the clustering of cells into islet-like aggregates in vitro
• high-level (127,000 cells/mL) - Cells cultured at this high-level inoculation had greater than double the percent of endocrine cells when compared to cells inoculated at a low-level and mid-level density after 9 days of culture -insulin-positive cells increased by 750%
• Cells cultured at a pH of 7.3 were found to have the highest percentage of endocrine cells
• cells cultured as aggregates were found to have almost 3 times higher cell densities- islet cells need to exist as aggregates in order to function properly
• The glucose stimulation and maximum stimulation indices are approximately 5.5 ± 0.6 and 39.9 ± 4.7 (ratio of the amount of C-peptide produced at 20 mM glucose plus 10 mM theophylline to the amount of C-peptide produced at 2.8 mM glucose)

Redwan, E. M., Matar, S. M., El‐Aziz, G. A., & Serour, E. A. (2007). Synthesis of the Human Insulin Gene: Protein Expression, Scaling Up and Bioactivity. Preparative Biochemistry & Biotechnology, 38(1), 24–39. https://doi.org/10.1080/10826060701774312

• The native insulin was generated by enzymatic conversion of chemically processed proinsulin.
• The produced insulin was matched with that of a commercial aqueous version at a level of enzyme immunoassys, SDS‐PAGE, RP‐HPLC, and bioactivity.
• The present results showed that the produced insulin has a comparable biochemical and potency similar to that of commercial one.

Sharp, J. and Vermette, P. (2017), An In-situ glucose-stimulated insulin secretion assay under perfusion bioreactor conditions. Biotechnol Progress, 33: 454-462. https://doi-org.proxy1.lib.uwo.ca/10.1002/btpr.2407

• Perfusion bioreactors offer stringent control of physiological parameters such as pH, flow, temperature, and dissolved oxygen concentration which have been shown to have an impact on cellular behaviour and viability
• Bioreacted cultures exposed to a high-glucose concentration showed the highest glucose-stimulated insulin secretion when compared to those in a low-glucose environment
• A lower incidence of apoptotic cells was observed in the bioreacted cultures when compared to non-bioreacted ones

Polez S, Origi D, Zahariev S, Guarnaccia C, Tisminetzky SG, Skoko N, Baralle M. A Simplified and Efficient Process for Insulin Production in Pichia pastoris. PLoS One. 2016 Dec 1;11(12):e0167207. doi: 10.1371/journal.pone.0167207. PMID: 27907132; PMCID: PMC5131935.

• Optimized all transpeptidation reaction conditions including temperature, reaction time, enzyme concentration, pH and concentration of organic solvents in order to improve the digestion conversion rate and to develop a cost-efficient process, reaching a 75% recovery.
• Second freeze-drying step was omitted whilst introducing an efficient and inexpensive crystallization step with no consequences on the final insulin product quality.

1. Amini, N., Paluh, J. L., Xie, Y., Saxena, V., & Sharfstein, S. T. (2020). Insulin production from hiPSC‐derived pancreatic cells in a novel wicking matrix bioreactor. Biotechnology and Bioengineering, 117(7), 2247-2261.
2. Braune, K., Lal, R. A., Petruželková, L., Scheiner, G., Winterdijk, P., Schmidt, S., ... & Hussain, S. (2022). Open-source automated insulin delivery: international consensus statement and practical guidance for health-care professionals. The Lancet Diabetes & Endocrinology, 10(1), 58-74.
3. Chawla, M., Bodnar, C. A., Sen, A., Kallos, M. S., & Behie, L. A. (2006). Production of islet‐like structures from neonatal porcine pancreatic tissue in suspension bioreactors. Biotechnology progress, 22(2), 561-567.
4. Gallegos, J. E., Boyer, C., Pauwels, E., Kaplan, W. A., & Peccoud, J. (2018). The Open Insulin Project: A case study for ‘biohacked’ medicines. Trends in biotechnology, 36(12), 1211-1218.
5. Magrofuoco, E., Elvassore, N., & Doyle III, F. J. (2012). Theoretical analysis of insulin‐dependent glucose uptake heterogeneity in 3D bioreactor cell culture. Biotechnology progress, 28(3), 833-845.
6. Mihara, Y., Matsuura, K., Sakamoto, Y., Okano, T., Kokudo, N., & Shimizu, T. (2017). Production of pancreatic progenitor cells from human induced pluripotent stem cells using a three-dimensional suspension bioreactor system. Journal of tissue engineering and regenerative medicine, 11(11), 3193–3201. https://doi.org/10.1002/term.2228