Abstract

Laboratory process automation is an important requirement for streamlining and standardizing technical procedures. Despite the extensive use of magnetic cell separation, only the latter steps in these procedures have been automated. Currently magnetic cell labeling is done manually followed by automated magnetic separation (e.g. AutoMACS and Isolex). Additionally, current technology only allows for processing of a single sample at a time. Our objective was to develop a fully automated system to magnetically separate multiple blood and bone marrow samples. The major barrier to automation of cell labeling is that these procedures typically require a centrifugal wash step, which is relatively expensive to automate and requires bulky equipment. We had previously developed a magnetic cell labeling/separation system call EasySep® (Stemcell Technologies) which does not require a centrifugal wash step. We have now fully automated EasySep® and present the RoboSep™ instrument which magnetically labels and separates 4 samples at once, with up to 2×109 total cells per sample or 8×109 total cells. The instrument operates in a standard biosafety hood and uses sterile disposable pipette tips to ensure aseptic operation and avoid cross-contamination between samples. Standardized automation protocols have been developed for both positive and negative selection. With positive selection, the desired cells are magnetically labeled and then purified by a sequence of magnetic wash steps. With negative selection, unwanted cells are magnetically labeled and then depleted. To demonstrate the suitability of RoboSep™ for automated positive selection of hematopoietic progenitors and stem cells, we performed CD34+ cell selection from previously frozen cord blood (CB) and mobilized peripheral blood (MPB). For the CB separations, the CD34+ cell content was enriched from 1.2±0.4% to 96.6±3.1% with a recovery of 45±9% (n=9, mean ± 1 SD). For the MPB separations the CD34+ cell content was enriched from 0.7±0.1% to 96.7±3.1%, with a recovery of 45±13% (n=4). To test RoboSep in negative selection we used an EasySep® antibody cocktail depleting cells that express any of CD2, CD3, CD11b, CD11c, CD14, CD16, CD19, CD24, CD56, CD66b, and glycophorin A to isolate hematopoietic progenitors from bone marrow (BM) and MPB. CB separations required the addition of anti-CD41 to the antibody cocktail for depletion of platelets. The table below shows results for negative selection from BM, CB and MPB. Manual separations performed in parallel with the above automated separations showed comparable purity and recovery, indicating that we have succeeded in automating both positive and negative selection procedures. The RoboSep instrument processes up to 4 tissue samples at once and provides the opportunity to isolate multiple cell subsets from the same sample by combining positive and negative selection methods in a single automated procedure.

Negative Selection Results (Mean± 1 SD)

Sample % CD34+ in start % CD34+ in enriched % Recovery CD34+ cells Fold-enrichment of total BFU-E, CFU-GM, CFU-GEMM % recovery of total BFU-E, CFU-GM, CFU-GEMM 
N.A. Not Available 
CB (n=2) 1.5 67.4 50 36 38 
MPB (n=2) 1.1 50.0 45 50 41 
BM (n=4) 4.7±3.1 47.5±7.5 N.A. 47±10 71±13 
Sample % CD34+ in start % CD34+ in enriched % Recovery CD34+ cells Fold-enrichment of total BFU-E, CFU-GM, CFU-GEMM % recovery of total BFU-E, CFU-GM, CFU-GEMM 
N.A. Not Available 
CB (n=2) 1.5 67.4 50 36 38 
MPB (n=2) 1.1 50.0 45 50 41 
BM (n=4) 4.7±3.1 47.5±7.5 N.A. 47±10 71±13 

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