Flexible Purecell Select System Enables Protocol Modifications to Optimize Enriched MNC Population for Downstream Applications

Transcription

1 Application Note PN3356 Flexible Purecell Select System Enables Protocol Modifications to Optimize Enriched MNC Population for Downstream Applications Introduction Pall s extensive knowledge and experience in blood cell interaction with materials and medical device development has resulted in a first-generation product named the Purecell Select System. Designed for mononuclear cell (MNC) recovery from whole blood (WB), the Purecell Select System is easy to use and produces consistently high MNC recovery in less than 15 minutes. Additionally, the Pall system has a sterile fluid path with very few points of direct user interaction. The Purecell Select System process is simple the sample is transferred to the sample bag via a sterile Luer-Lok port, then as the sample flows through the system under gravity flow, the MNC are captured on Pall s filter materials. After flow has stopped, the user back-flushes the filter with the optimized Harvest Solution to recover the enriched MNC population. Purecell Select System processing results in a sample with a significant reduction in total red blood cells (RBC), platelets, and volume. Pall recognizes that for some applications the standard performance is not optimal for downstream use. Simple modifications to the standard protocol can significantly alter the cellular composition of the final product. This Application Note is intended to provide useful information on the performance of this system when the standard protocol is modified. Results from WB starting volume of 2-12 ml, Harvest Solution volumes of 1-25 ml, the inclusion of a rinse step, and fresh versus 24-, 48-, and 72-hour WB samples are presented. Additionally, recommendations for post-processing sample manipulation for RBC removal, centrifugation, and freeze-thaw are included. Materials and Methods Human WB Samples Units of human whole blood collected in CPDA-1 anticoagulant are held at ambient temperature until use. Experiments are performed within three hours of blood collection, except for WB 1, 2, or 3 days after collection, as noted. Mononuclear Cell Enrichment by Filtration (8-15 minutes total processing time. Refer to instructions for use for labeled diagram and full details.) A syringe is used to transfer WB and approximately 1 ml of air into the harvest set. 5 ml WB is used for all experiments unless noted. The WB passes through the Purecell Select System by gravity flow. Cells are recovered by back-flushing (i.e., reversing the direction of the flow) with 24 ml sterile Purecell Select System Harvest Solution, unless noted otherwise in figure legend. Incorporation of the optional rinse is performed in the following manner: Close the upper clamp to stop WB flow once the WB sample reaches the mid-point of the Y at Port A (sample input port). Attach a syringe filled with pre-measured rinse solution [Phosphate Buffered Saline (PBS) for these tests] to Port A and add slowly. Be sure that there are no air bubbles in the tubing between the blood and the rinse solution. Any air bubbles can be removed by squeezing the tubing to force the bubbles upward, or by flicking or tapping the tubing. Open the clamp to restart the sample flow. All Purecell Select System processing procedures were done on an open lab bench using good laboratory practice, not in a laminar flow biological safety cabinet.

2 Materials and Methods (continued) Cell Counts Generated on a Cell-Dyn 18 hematology analyzer (Abbott Labs) following standard protocols. Triplicate measurements are averaged for the concentrations of WBC (white blood cells), RBC, and platelets. Percent recoveries for each sub-population are determined by calculating the number of cells before and after the preparation using the following formula: Concentration of cells after isolation procedure x volume Concentration of cells before isolation procedure x WB volume x 1 Three-Part Differential Based on WBC counts from Cell-Dyn analyzer and lymphocyte, monocyte, and granulocyte percentages from forward versus side scatter plots on a BD FACSCalibur flow cytometer (BD Biosciences) unless noted in figure legend. MNC numbers are a sum of lymphocytes and monocytes. The formula used is as follows: (Concentration of WBC x volume x percent of population) / 1 Viability Determination Percent viability of WBC in the starting material and after processing is determined by dye exclusion using propidium iodide (PI). 5 µl sample, pre- and post-filtration, is incubated in 1 ml 1x H-lyse buffer (R&D Systems) for 18 minutes at RT. 1 or 2 µl of PI (1 mg/ml, Molecular Probes) is added (except no stain controls) and incubated an additional 2 minutes at RT. The cells are pelleted by centrifugation at 5 x g for 5 minutes, then resuspended in.5 ml PBS for analysis. Percent viability is determined by calculating the percentage of unstained versus total cells as determined by flow cytometry (BD FACSCalibur). RBC Depletion for Colony Forming Unit (CFU) Assays RBC depletion prior to cell culture is recommended. NH 4Cl solution (StemCell Technologies) is used with slight modifications to manufacturer s protocol. 5 ml of Purecell Select System processed cells are added to 2 ml of ice-cold NH 4Cl solution (final ratio is 4:1 lysis buffer:sample). The mixture is incubated on ice until lysis is complete (~1-15 minutes). After complete lysis, 25 ml of PBS with 2% fetal bovine serum (FBS) is added. Then WBC cells are centrifuged at 5 x g for 1 minutes. The cell pellet is resuspended in 25 ml of PBS with 2% FBS and centrifuged again at 5 x g for 1 minutes. The final cell pellet is resuspended in Iscove s Medium with 2% FBS. CFU Assays (StemCell Technologies) Performed according to manufacturer s instructions. Processed samples are resuspended at a concentration of 2 x 1 6 WBC/mL. 3 µl of cell suspension is added to 3 ml of complete Methocult. 1.1 ml of Methocult-cell suspension (2 x 1 5 WBC/dish) is added to each of two 35 mm culture dishes. Culture dishes are transferred to a tissue culture incubator at 37 ºC, 5% CO 2, and 95% humidity. The CFUs are enumerated after days in culture. Enumeration of CFU The CFU is counted using an inverted microscope (Olympus 1x51). Cultures are evaluated for the presence of erythroid, myeloid, and multi-potential CFU as described in the technical manual, Human Colony-Forming Cell Assays Using Methocult (Stem Cell Technologies). Erythroid CFC include Burst-Forming Units-Erythoid (BFU-E) and Colony-Forming Unit-Erythroid (CFU-E). The Myeloid CFC include Colony-Forming Unit-Granulocyte (CFU-G), Colony-Forming Unit-Macrophage (CFU-M), and Colony-Forming Unit-Granulocyte, Macrophages (CFU-GM). Multi-potential CFC include Colony-Forming Units with mixed populations of erythroid and myeloid cells (CFU-GEMM). Results WB Volume Testing Purecell Select System Results Using 2-12 ml of WB The bar graph in Figure 1 shows total number of MNC and granulocytes (left and right, respectively) and percent recovery from the Purecell Select System processing of 2, 4, 8, and 12 ml of human WB. As expected, the number of MNC and granulocytes in the Purecell Select System sample increases with increasing volume of WB (blue bars). There is a slight decrease in the percent recovery of MNC as the WB volume increases, as seen by the increasing gap between cell number in the WB sample (orange bars) and the Purecell Select System sample (blue bars) at higher volumes. Interestingly, there is a dramatic decrease in granulocyte recovery as the WB volume increases. This results in a relatively greater enrichment of MNC as compared to granulocytes with increasing volume. When the volume is increased to 15 ml, there is a significant reduction in percent MNC recovery (data not shown). Thus, 12 ml is the upper limit recommended for this filter harvest set design.

3 Results (continued) Figure 1 Purecell Select System Total Cell Number and Recoveries from 2-12 ml of WB Total Cell Number (x (x 1 6 ) ) % 85% 77% 71% 79% 55% 38% 31% Mononuclear Cells Granulocytes WB Purecell Select System Total number and percentage of mononuclear cells and granulocytes in WB or Purecell Select System from indicated volume of WB (2, 4, 8, 12 ml). Samples processed in duplicate. 2 ml volume from 3 donors; all others from 5 donors. Recovery is calculated based on WBC and three-part differential from Cell-Dyn 18 hematology instrument. Error bars show one standard deviation (SD) across donors. Harvest Solution Volume Testing Purecell Select System Results Using 1-25 ml of Harvest Solution (Figure 2 and Table 1) The bar graph in Figure 2 shows the percent recovery of MNC, lymphocytes, monocytes, and granulocytes from the Purecell Select System processing when 1, 15, 2, and 25 ml of Harvest Solution are used for the back-flush step. As expected, increasing the volume of Harvest Solution results in higher recovery of the major populations. The difference in recovery is ~6-1% of the starting cell number when comparing 1 ml to 25 ml. Although cell recovery is lower with less Harvest Solution, the concentrations of MNC and granulocytes are significantly higher when lower volumes of Harvest Solution are used (Table 1). MNC concentration drops from a 4.4 fold increase to a 1.6 fold increase relative to WB with 1 versus 25 ml Harvest Solution, respectively. Thus, there is a trade-off between MNC recovery and final cell concentration. This can be countered by processing a higher volume of WB. The relative ratio of MNC compared to granulocytes changes somewhat with the volume of Harvest Solution. Figure 2 Percent Recovery of Major Cell Populations with 1, 15, 2, or 25 ml of Harvest Solution Percent Recovery MNC Lymph Mono Gran RBC Plts 1 ml 15 ml 2 ml 25 ml Standard protocol used except that the volume of Harvest Solution for cell recovery is varied, as indicated. Percent recovery is calculated as described in Materials and Methods. Duplicates are averaged. Means +/- 1 SD across 3 donors are reported. Table 1 Effect on Final MNC and Granulocyte Concentration of 1, 15, 2, or 25 ml of Harvest Solution Average Fold Increase (x 1 6 Cells/mL) (x 1 6 Cells/mL) Average Final MNC Gran MNC Gran Volume (ml) WB ml ml ml ml Data generation (3 donors) and analysis as described in Figure 2 legend. Optional Rinse Step to Further Reduce RBC and Platelets For greater reduction in the number of RBC and platelets, an additional rinse step is incorporated into the processing protocol. Specifically, WB flow is stopped once the level of WB has reached the sample input valve. PBS is then added to the system through the sample input valve. The flow is resumed and the processing is continued as per the standard method (see Materials and Methods). The recovery of the major cell types is shown in Figure 3a. The standard Purecell Select System method results in 86% depletion of RBC. When an additional 6 or 1 ml PBS rinse is incorporated, RBC depletion is increased to 94% and 97%, respectively. The rinse step also results in a 5-8% further reduction in platelets. There is a 5-2% decrease in recovery of lymphocytes, monocytes, and granulocytes with the added rinse step. Lymphocytes, which typically give the highest recovery, show the greatest drop in recovery with this step. The viability of the processed samples is similar or slightly improved with the additional rinse step (data not shown).

4 Results (continued) Figure 3a Effect of a Rinse Step on Recovery of Major Cell Populations 5 Percent Total Recovery Cell Number (x 1 6 ) % 85% 77% 71% 79% 55% 38% 31% Mononuclear Cells Granulocytes WB Purecell Select System MNC Lymph Mono Gran RBC Plts No rinse 6 ml rinse 1 ml rinse The standard protocol +/- the optional PBS rinse step is followed, as indicated on the graph. Means +/- 1 SD across 3 donors are reported. Data from rare cell enumeration using CFU assays for erythroid, myeloid, and mixed colonies demonstrate the presence of hematopoietic progenitor cells in WB samples processed using the Purecell Select System (Figure 3b). Although the level of RBC in the harvested sample is much lower than the starting material, further depletion is necessary before plating to achieve accurate colony counts. Data comparing the Purecell Select System rinse protocol versus the standard protocol with RBC lysis suggests that some of the erythroid progenitor cells are lost during the RBC lysis and/or subsequent washing step. The PBS rinse step saves time while resulting in equivalent or higher numbers of hematopoietic progenitor cells. Thus, even though the rinse step does result in a ~5-2% decrease in total MNC recovery, the rare cells do not appear adversely effected when compared to the incorporation of an RBC lysis step. Figure 3b Effect of a Rinse Step on Hematopoietic CFU Assays CFU Number/2 x Erythroid WB RBC Lyse Pall RBC Lyse Myeloid CFU Type Pall 6 ml Rinse Mixed Pall 1 ml Rinse CFU assay results from samples processed as indicated. The standard method sample required an RBC lysis step prior to use in this assay, while samples processed with the additional rinse step were diluted and used directly in the assay. Duplicates are averaged. Means +/- 1 SD across 3 donors are reported. Purecell Select System Processing of Fresh Versus 1, 2, or 3-Day Old WB Recovery and viability data from WB pre- and post-processing with the Purecell Select System (Table 2) indicates that fresh and aged WB samples (from 3 donors) behave similarly. There are no significant differences in percent recovery of the MNC, WBC, or cell composition of the samples (data not shown) between WB processed on the day of collection or 1-3 days after collection when compared to pre-processing values. There may be a slight decrease over time in total MNC and WBC number, but donor variability is high for these counts. The viability of major cell populations post-processing is always equal to or slightly higher than the WB starting sample of the same age. There is a slight downward trend in WB viability over time, which is to be expected. Table 2 Purecell Select System Processing of Fresh WB Compared to Aged WB Percent Recovery and Viability Cell Number x 1 6 /5 ml WB % Recovery or Processed Sample WB Age Sample MNC Recovery WBC Recovery MNC WBC Viability Fresh WB / / /- 9. Pall 74. +/ / / / /- 5.7 Day 1 WB / / / Pall / / / / /- 7.1 Day 2 WB / / /- 5.9 Pall / / / / /- 3.9 Day 3 WB / / /- 3.2 Pall / / / / /- 4.6 Comparison of MNC and total WBC recovery and viability from matched donors using fresh WB versus the same WB stored at 4 ºC for 1-3 days. Chilled WB is equilibrated to room temperature prior to processing. Percent viability is determined with PI. Averages from 3 donors +/- 1 SD are reported.

5 Recommendations for Centrifugation of Purecell Select System Processed Samples Although isotonic, the Harvest Solution has higher density than typical buffers. As a result, good recovery from a centrifugation step requires the use of non-standard conditions. Pellet the cells at 3-4 x g for 5 minutes with no brake. It is crucial to turn off the centrifuge brake! If this is not done, the cell pellet will be highly disrupted, resulting in poor recovery. Resuspend the cell pellet in appropriate buffer. This procedure will result in a significant decrease in platelets (4-75% relative to pre-centrifugation), but high recovery of all other cell types. Cell Freeze Recommendations for Samples Processed Using the Purecell Select System Several factors such as cell concentration, solutions, and serum proteins used for preparation of cell suspension, concentration of cryopreservation agent, cooling rate, storage conditions, and thawing methods directly affect the viability and quality of cells after freezing and thawing. Freeze and thaw procedures should be optimized for the MNC samples and any post-thaw manipulation step. RBC lysis during a freeze-thaw cycle contributes to post-thaw debris in any RBC-containing sample. If desired, RBC may be removed prior to cell freezing using an RBC lysis method or by adding a rinse step to the Purecell Select System standard processing protocol after cell capture and before cell harvest (see note above regarding rinse step). If an RBC lysis step is included, be aware that the number of RBC in the processed sample will be much lower as compared to WB (< 15% of total from 5 ml WB). We typically use half the amount of RBC lysis solution recommended for WB samples when performing RBC lysis of Purecell Select System processed samples. During the post-rbc lysis wash and centrifugation, turn off the brake (as noted above) unless sample dilution is 1-fold or greater. Post-thaw viability determination of MNC or specific target cell population is recommended. Analysis of thawed samples indicates that a high percentage of the granulocytes (CD66b positive cells) die during freeze-thaw, as assessed with PI staining (data not shown). This is consistent with the well-known fact that granulocytes do not survive freeze thaw cycles as well as other cell types. As a result, postthaw viability on total WBC is not indicative of MNC viability. Conclusions Pall s Purecell Select System provides fast sample processing with an easy-to-use method resulting in high MNC recovery. In addition to use of the standard protocol, simple alterations to the procedure change the final MNC sample composition. The system is very robust and flexible, readily accommodating WB volumes from 2-12 ml, Harvest Solution volumes of 1-25 ml, and an added rinse step to reduce RBC or older WB samples (fresh to 3 days). Guidelines are provided for post-processing sample manipulation, specifically cell freezing and centrifugation. Matched donor samples are used for all testing to eliminate donor-to-donor variability, which is very high. Most experiments are done with 5 ml volumes from split blood units and data from multiple donors is combined, although generally with a limited number of samples. Data from duplicates shows a high degree of processing method reproducibility, consistent with overall high reproducibility reported in Application Note Performance Characterization of the Purecell Select System for Enrichment of Mononuclear Cells from Human Whole Blood (PN 3355). Although it is unlikely that the Purecell Select System will replace Ficoll cell processing for all applications, the ability to modify the protocol to alter final sample composition is beneficial to users. This method flexibility increases the range of downstream applications for which the Purecell Select System processing method is directly applicable.

6 Pall Medical - Cell Therapy 6 South Wagner Road Ann Arbor, MI USA toll-free in USA phone fax Stem_Cells@Pall.com Australia - Cheltenham, VIC Tel: (in Australia) Fax: Brazil - Rio de Janeiro Tel: (5521) Fax: (5521) Canada - Ontario Tel: (in Canada) Fax: Canada - Québec Tel: (in Canada) Germany - Dreieich Tel: Fax: India - Mumbai Tel: or Fax: Korea - Seoul Tel: Fax: Thailand - Bangkok Tel: (66) Fax: (66) United Kingdom - Portsmouth Tel: +44 () Fax: +44 () Biosvc@Pall.com United States - California Tel: Fax: Stem_Cells@Pall.com 28, Pall Corporation. Pall,, and Purecell are trademarks of Pall Corporation. indicates a trademark registered in the USA. Ficoll is a trademark of GE Healthcare Bio-Sciences. Cell-Dyn is a trademark of Abbott Laboratories. BD FACSCalibur is a trademark of BD Biosciences. Luer-Lok is a trademark of Becton-Dickinson. This product, and its use, may be covered by one or more patents including US 6,544,751 and EP 973,587. 1/9, 1.5k, GN8.234 PN3356