Evaluation of the Impact of a New Cooling Cell Processor System on Islet Cell Isolation Facility

Summary

The protocol here describes a custom cooling cell processor system that is compatible with a good manufacturing practices (GMP) cleanroom to improve cell viability post-processing.

Abstract

Standard cell therapy equipment, including the gold standard cell processor to purify human islets for clinical transplantation, is rarely refrigerated, potentially exposing cells to elevated temperatures during the centrifugation step. Custom cooling systems have a direct benefit on human islet viability and function. The current study was designed to test the effectiveness of a newly developed, readily available cooled cell processor system requiring minimal modifications and to evaluate its impact on human cell viability and the GMP cleanroom environment. The cooler system, a mechanically refrigerated heat exchanger set at -30 °C was used to deliver cooled medical grade dry air to the cell processor bowl through a hole drilled in the centrifuge cover. With the limited availability of pancreas donors in Qatar, system validation was done with continuous density gradient purification of pooled human bone marrow buffy coat. Sterility, turbulence, and particle count were measured in class C and class B clean room environments. No turbulence developed around the cooled cell processor, and no excess 0.5 µm and 5 µm airborne particulates were generated as per cleanroom GMP standards. At the beginning and end of the collection steps, the temperature rose respectively to 21.50 °C ± 0.34 °C and 21.93 °C ± 0.20 °C in the non-cooled cell processor and to only 10.9 °C ± 0.17 °C and 11.16 °C ± 0.35 °C in the cooled- cell processor (p <0.05). The cooled cell processor led to both improved recovery (98%) of the mononuclear cell fraction and viability (100% ± 2%) post-processing. The new cooling system effectively reduces the heat produced by the cell processor while having no particulate impact on the GMP clean room environment. The cooled cell processor described here is an inexpensive ($16,000 without including taxes, customs clearance, and transportation) and minimally invasive method to provide robust cooling. Currently, this technology in the GMP cell therapy facility is being applied to human islet cell isolation and transplantation for the clinical program.

Introduction

Islet transplantation for severe Type 1 diabetes can restore endogenous insulin secretion and has become the standard of care in a number of countries¹. Islet isolation is a multi-step procedure that includes warm enzymatic and mechanical digestion of the pancreas, followed by cooled phases for recovery of digested tissue and islet purification with continuous or discontinuous density gradients on a cell processor. Rapid cooling slows the enzymatic digestion phase and protects islets from digestive enzymes liberated by contaminating acinar cells. The cell processor was originally designed to separate cellular blood components at ambient temperature². It was repurposed to purify human pancreatic islets of Langerhans in 1989³. For three decades, human islets have been purified for clinical trials and, more recently, standard-of-care, either in a cell processor with discontinuous gradients⁴ or continuous density gradients¹ made with a gradient maker¹ or equivalent computer-operated mixer^5,6,7. Other groups have reported islet purification from large mammals and humans using large cylindrical plastic bottles^8,7,9, which substantially improved the efficacy of purification and islet quality.

Indeed, considering the gold standard to purify human islets for clinical transplantation^10,11, the manufactured cell processor was without refrigeration. As a direct consequence of the lack of refrigeration, islets were exposed to increasing temperatures during the purification step. During centrifugation, an uncontrolled increase in temperature occurs due to the mechanical friction of the rotor^12,13. Cell processors were adapted with a cooling system in islet isolation centers that statistically increased glucose-induced insulin secretion and cell viability, confirming the advantage of keeping pancreatic islets at low temperatures during the purification procedure^14,15.

It should be noted that in the late 1980s, 10 refrigerated prototypes were built to reduce islet damage during density gradient purification but received no further attention. The cell processor prototypes were based on spindle and bowl cooling with chilled polyethylene glycol. In particular, islet purification solutions, including organ preservation solutions and polysaccharide density gradients, may be deleterious for islet viability when used at or above room temperature^16,17,18. A comparison of different cooling strategies of the cell processor for islet cell purification versus the original non-refrigerated cell processor was evaluated to combat the uncontrolled increase of temperature that occurs due to the friction of the rotor. Several attempts have been made to cool down the cell processor during human islet purification. The cell processor can be temporarily or permanently housed in a specially designed cold room to maintain a temperature of 0-8 °C¹⁹ during the gradient centrifugation which is currently used at the islet isolation centers at the University of Illinois Chicago¹⁹, University of Miami¹⁴, University of Oxford facilities¹⁵, and the University of San Francisco (the custom cooling unit encases the cell processor). Another modification includes core cooling of the cell processor spindle with a cooling system using electronically controlled liquid nitrogen cooling in Geneva, Sweden, and the Czech Republic^15,19. Another alternative in the mid of 2000 was the core cooling of the cell processor with an air-conditioning unit¹² or by liquid cooling of the shaft at the University of Lille.

Cooling modifications of the cell processor are not readily available and come with risks (humidity, condensation build-up) inside GMP cleanroom environments, including loss of warranty and refusal of the constructor to service the machine. Consequently, some islet centers, including Milan, Italy, Oslo, Norway, and Edinburgh, Scotland, do not use a cooling system for cell processors during the islet purification process¹⁵.

The current study was designed to test the effectiveness of a newly developed readily available cooler in conjunction with a minimally modified cell processor system. Briefly, the air-based cooler described here is a mechanically refrigerated heat exchanger controlling the temperature of 1 standard cubic foot per minute (SCFM) of dry gas between -40 °C to -100 °C. This cooling system requires a dry medical air supply. The air will flow through the line and heat exchanger and out through the (7.9 mm) nozzle. A heater located within the nozzle supplies heat to control the temperature of the air stream. A spare solid plastic centrifuge cover was purchased, and minimal modifications were made. A hole of 13.3 mm diameter was drilled in the cover, and cooled air was delivered from the cooler nozzle to the cell processor via the hole in the centrifuge cover. The cooled medical air was injected inside the centrifugation bowl of the cell processor with constant control of the temperature of the liquids or density gradients.

We evaluated the technical performance of the cooled cell processor and the impact of the cooling element on the GMP cleanroom environment. The main focus was to achieve a robustly low (<10 °C) temperature during human islet purification to preserve viability. Using a buffy coat density separation as a proxy for islet isolation, we determined the efficacy of this cooling protocol on cell viability.

Protocol

This protocol used discarded whole blood not fit for use as per institution discard guidelines. IRB approval and informed consent are not applicable. 1. Cooled cell processor setup NOTE: All procedures must be done in a clean room environment, and staff must respect clean-room garment procedures. Purchasing of equipment Purchase a spare plexiglass lid. Purchase the cooling equipment system, cooler with mechanically refrigerat…

Representative Results

The impact of the cooled cell processor on the GMP cleanroom was first quantified. Standardized methods used a particle counter measuring the airborne particle contamination in a GMP grade class C and class B clean room environment at critical points in proximity to the cell processor and pressurized air cooler system. Results demonstrated that no turbulence developed around the equipment. Recorded data showed that there was no excess of 0.5 µm and 5 µm airborne particulate as per cleanroom GMP grade C, grade B…

Discussion

Islet isolation facilities around the world have adopted for decades human islet purification in a repurposed cell processor. Although some islet production facilities continue to process islets in an unrefrigerated cell processor, most centers use a refrigerated version of the cell processor^10,11,15. Centers either place the cell processor in a cold room which has the major disadvantage of exposure to humidity and condensa…

Materials

Equipment
Air particles counter	Lasair III
AirJet XR40	SP Scientific	FTS system XR40
Biosafety Cabinet	Thermo Scientific	EQ-1301
Circulating Cooling Chiller	Julabo	CF30
COBE 2991	Terumo BCT
Conical Cooling Tray	Biorep	CCT-01
Double jacketed gradient maker	customized in house
KJT-Thermocouple Thermometer	Hanna Instruments	HI93551N	For measuring liquids temperature
Magnetic stirrer	Thermo Scientific	88880014
Peristaltic pump	MasterFlex	MK-77921-79
Thermometer	Extech Instruments	RMS 430	For COBE temperature
Waterless Bead Bath	Cole-Parmer	10122-00
Materials and reagents
BD stem cell enumeration kit	BD Biosciences	344563
COBE 2911 tubing kit	Terumo BCT	90819
Conical Tubes 250ml	Corning	430776
Gradient density 1.1	Biochrom	L6155
Graduated disposable bottles	Thermo fisher	382019-1000
Human albumin 20%	Kedrion Biopharma	8091600
M199 washing solution	Corning	99-784-CM
Masterflex tubes	Masterflex	96400-6
Medical Dry Air	Linde Healthcare	Medi On 22
Pencillin-streptomycin	Gibco	15140122
Thermoprobe	Biorep	TC-02	Thermosensor
Trypan blue	Sigma Aldrich	15250061
University of Wisconsin Belzer (UW)	Bridge of Life	RM/N 4055	Conservation Medium