Automated Counterflow Centrifugal System for Small-Scale Cell Processing

Summary

Automation is key to upscaling and cost management in cell manufacturing. This manuscript describes the use of a counterflow centrifugal cell processing device for automating the buffer exchange and cell concentration steps for small-scale bioprocessing.

Abstract

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and product concentration are essential components for most manufacturing processes. However, at the early stages of product development, these steps are often performed manually. Manual dead-end centrifugation for buffer exchange is labor-intensive, costly, and not scalable. A closed automated system can effectively eliminate this laborious step, but implementation can be challenging. Here, we describe a newly developed cell processing device that is suitable for small- to medium-scale cell processing and aims to bridge the gap between manual processing and large-scale automation. This protocol can be easily applied to various cell types and processes by modifying the flow rate and centrifugation speed. Our protocol demonstrated high cell recovery with shorter processing times in comparison to the manual process. Cells recovered from the automated process also maintained their proliferation rates. The device can be applied as a modular component in a closed manufacturing process to accommodate steps such as buffer exchange, cell formulation, and cryopreservation.

Introduction

The landscape of modern medicine has transformed rapidly through recent developments in gene and cell-based therapies (GCT). As one of the fastest growing fields in translational research, the GCT sector also faces unique and unprecedented challenges. In addition to robust clinical outcomes, efficient and cost-effective manufacturing processes are essential for the commercial success of GCT, which is particularly difficult to achieve in small-scale manufacturing¹. The cost of time, labor, and quality assurances are magnified when each batch of cells only produces a few doses for one patient instead of hundreds or thousands. Unlike allogeneic ce....

Protocol

1. Preparation of reagents and cells for buffer exchange

1. Prepare buffers (see Table of Materials) in a class 2 laminar flow hood. Using a syringe and needle assembly, remove 50 mL of saline solution from a 500 mL saline bag. Replace this with 50 mL of 20% human serum albumin (HSA) to make 2% HSA in saline, which will serve as the wash buffer.
2. Remove the cells from the culture vessels and perform a cell count to determine the starting cell quantity and viability by trypan blue exclusion. Load the cells into a transfer bag.
   NOTE: In this protocol Jurkat cells were cultured in RPMI and MSCs were cultured in DMEM:F12. Both med....

Results

In this protocol, we used Jurkat cells and MSCs as representative examples to demonstrate the automated buffer exchange process. During the process, Jurkat cells and MSCs shared the same processing steps with differences in centrifugal force and pump speed that control the flow rate (Table 1). Figure 2 shows representative images captured by the camera of how the fluidized cell bed may appear during the buffer exchange process. Typically, the fluidized cell bed will resemble.......

Discussion

The automated buffer exchange protocol described is simple and user friendly. Nevertheless, there are a few key steps in this protocol that are critical and require particular attention. In our experience, when processing larger cells such as MSCs (average diameter 10–15 µm) each run should include at least 1 x 10⁷ cells to achieve optimal cell recovery (Figure 4B). Processing smaller cells, such as Jurkat cells (average ~10 µm diameter), requires .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
20 ml Luer lock syringes	BD	302830
20% Human serum albumin (HSA)	CSL Behring	AUST R 46283
4-(Dimethylamino)benzaldehyde	Sigma-Aldrich	156477-25g
500ml IV saline bag	Fresenius Kabi	K690521
Antibiotic-Antimycotic	Thermo Fisher Scientific	15240112
Automated cell counter (Countess)	Thermo Fisher Scientific	N/A
Cell counting chamber slides	Thermo Fisher Scientific	C10228
Cell stimulation cocktail (500x)	Thermo Fisher Scientific	00-4970-93
Cell transfer bags	Terumo	T1BBT060CBB
CellTiter AQueous One Solution Cell Proliferation Assay (MTS)	Promega	G3582
Centrifuge	Eppendorf	5810R
DMEM: F12 media	Thermo Fisher Scientific	11320082
EnVision plate Reader	Perkin Elmer	N/A
Fetal bovine serum (FBS)	Thermo Fisher Scientific	10099141
Human Interleukin 2 (IL2) Kit	Perkin Elmer	Al221C
Luer (female) fittings	CPC	LF41
PC laptop or PC tablet device	ASUS	N/A
Plate reader (SpectraMax i3)	Molecular Device	N/A
Recombinant Human IFN-γ	PeproTech	300-02
Rotea counterflow centrifuge cell processing device	Scinogy	N/A
Rotea single-use processing kit	Scinogy	N/A
RPMI media	Thermo Fisher Scientific	11875119
Surgical scissors	ProSciTech	420SS
Trichloroacetic acide	Sigma-Aldrich	T6399-250g
Trypan Blue stain	Thermo Fisher Scientific	T10282
Trypsin digestion enzyme (TrypLE Express Enzyme)	Thermo Fisher Scientific	12604013