STEMCLEAR© MEDIUM
StemClear is a density gradient made of silane coated,
colloidal silica particles in an isotonic salt solution (PBS),
with a density of 1.080 g/mL and endotoxin levels below 0.25EU/mL. The purpose of the density gradient is to separate blood plasma and erythrocytes from leucocytes, through a single centrifugation step. The white blood cells can either be harvested and cryopreserved or further purified to collect the CD34+ stem cells.

Product features
StemClear has been developed and validated for use with cord blood, bone marrow and periferal blood. Validation in an independent laboratory has shown that white blood cell recovery is between 65 and 100% after density gradient centrifugation with StemClear.
StemClear is manufactured in accordance with GMP guidelines for maximum product safety and highest quality. StemClear is CE marked as a Class 2a medical device which makes it ready for use in any laboratory that has to comply with the latest European tissue directives.

Validation results
Purpose of the validation
The expertise group CTS (Stem cell laboratory, UMC St Radboud, Nijmegen, The Netherlands) regularly performs gradient separations for the enrichment of haematopoietic progenitor cells (HPC). The processed cells are used for stem cell transplants in human patients. The gradient system used by the CTS is of clinical grade. Until recently gradient separations were performed using a non-CE marked colloidal silica medium (hereafter called medium x) from a Scandinavian manufacturer. Due to the inconvenient packaging of the product (large volume, inconvenient bottle type) and the fact that the density of the medium had to be adjusted manually, the lab has been looking for a replacement product.
The purpose of this study is to test the usability of two media: StemClear and Optiprep as a replacement for medium x.

Test principle
The usability of StemClear and Optiprep as a replacement product for medium x for the enrichment of HPC for stem cell transplants, was tested by performing density gradient separations using gradient density of 1.080 g/mL.
The standard technique uses transfer bags containing the cell source (untreated blood). The density gradient medium is injected into these transfer bags, under the cell source. After the proper centrifugation cycle, the mononuclear cells (also containing HPC) are concentrated in a specific area in the density gradient (inter-phase), most of the erythrocytes can be found in the pellet (bottom) of the transfer bags.
By means of cell counting methods, cell cultivation techniques and flow-cytometric determination of the CD34+ cells, the difference of the efficacy and efficiency of the techniques can be determined.

Tests performed
Toxicity test on Bone Marrow (BM)
BM inter-phase cells that where HPC-enriched by density gradient centrifugation with the test media, where cultivated either in standard culture medium (reference value), or in cell culture medium diluted 1/1 with test medium. After 4 hours exposure the cells are cultivated using standard HPC cultivation methods.

Toxicity test on Peripherous blood Stem Cells (PSC)
PSC cells are exposed for 4 hours in standard culture medium (IMDM = reference), and the test media. After the 4 hours exposure the cells are tested using standard HPC cultivation methods and treated n the same way as described in the toxicity test on Bone Marrow.

Density gradient separation in tubes using peripherous blood to determine the separation layers into discreet cell populations in the gradient
Peripherous blood was evenly distributed into 3 tubes, the cell solutions where diluted with cell culture medium until the total volume in the tube was 35mL. 11mL of test medium was then injected into the same tube, below the cell/medium mixture. The tubes where centrifuged and after centrifugation the inter-phases where collected and washed using HBSS medium and diluted in FSC.

Density gradient separation in tubes on BM, PSCA and cord blood in order to determine which density gradient
is most suited (as compared to medium x) for use with transfer bags
Bone marrow, cord blood and peripherous stem cells where divided into 3 test tubes, these cell solutions where diluted with cell culture medium until the volume per test tube was 35mL. 11mL of test medium was then injected into the same tube, below the cell/medium mixture. The tubes where centrifuged. After centrifugation the inter-phases where collected and washed using HBSS medium and diluted in FSC. The cell recovery is measured using the Coulter ACT10 cell counter; the subsets of the cells are determined using immune-phenotyping.

Density gradient separation in transfer bags using BM in order to determine the separation characteristics
of the replacement media compared to medium x separation
Density gradient separation in transfer bags was performed on 3 bone marrow aspirates for use in bone marrow transplants. Of 1 transfer bag of bone marrow, one halve was prepared using medium x and the other halve using a replacement density gradient.

Density gradient separation in transfer bags using cord blood in order to determine the separation characteristics
of the alternative media
Density gradient separation in transfer bags was performed on cord blood; the results where compared to density gradient separation using medium x in tubes. This technique was used due to the limited volume of the cord blood samples.

Conclusion of the tests
StemClear is a valid alternative to existing media for density gradient separation of bone marrow and for cord blood transplants.

Clinical results
In total 19 allogenous bone marrow transplants have been performed using bone marrow treated by density gradient separation with StemClear. The re-population of the patients receiving such a transplant are comparable to those obtained after medium x or Percoll separation. No adverse events related to the use of the density gradient were observed in the initial patient group.

Product specifications
StemClear undergoes rigorous quality control before being released. Each batch is manufactured to the following specifications
• pH: 7.15 – 7.40
• Osmolality: 295-310 mOsm/kg
• Sterility: SAL 10^-3
• Density: 1.080-1.082 g/mL
• Endotoxin: < 0.25 EU/mL
A certificate of analysis is available on request.

Zuletzt geändert am: Jun 24 2011 um 10:00 AM