APPLIED BIOLOGICS

Osteoarthritis

AUTOLOGOUS STEM CELL CONCENTRATION AT POINT OF CARE

+*Jacobson, M.S.; +*Kevy, S.V.; **+Mandle, R.J.

*Children’s Hospital, Harvard Medical School; +The CBR Institute for Biomedical Research

**Bioscience Research Associates, Boston, MA

Email:  jacobson@cbr.med.harvard.edu

Introduction:

Bone marrow stromal cells (BMCs) are pleuripotent adult stem cells that have been shown to differentiate into different cellular phenotypes.  In this study, we evaluated a methodology to concentrate bone marrow (BMC) at point of care within 15 minutes of collection.

 

Methods:

Sixty (60) mL of bone marrow was withdrawn with a single aspiration from the posterior iliac crest into a syringe containing preservative free heparin (20-50 U heparin per mL of bone marrow).  The procedure had IRB approval; all donors were tested for transmissible diseases, and were financially compensated.  The concentration process utilized the Harvest Technology (Plymouth, MA) Smart PReP2 system that contains a modified floating shelf of a specific density in the first chamber of the processing disposable which allows the nucleated marrow cells and platelets to rise above the shelf during the initial centrifugation.  These are then decanted into the second chamber of the disposable and concentrated.  The system can process two bone marrow samples, or concomitantly 60 mL of whole blood for the preparation of platelet gel or utilize a designed counter balance.  The bone marrow aspirates were analyzed numerically and morphologically pre and post concentration.  Hematopoietic stem cells (CD34+) were used to evaluate bone marrow stem cell concentration because of their higher frequency as compared to mesenchymal stem cells (MSCs).  The process of bone marrow derived stem cell isolation and expansion was evaluated utilizing specifically formulated media supplements to differentiate into osteogenic and chondrogenic lineages.

 

Results:

A summary of the results obtained preparing bone marrow concentrates in our laboratory and at point of care, are shown in Tables 1 and 2.

 

Characterization of Bone Marrow Concentrate

 

Table 1:  MEAN VALUES:  N = 16 volunteer donors from commercial source

Total TNC x 106 BMA Total TNC x 106 BMC Total MNC x 106 BMA Total MNC x 106 BMC CD34+ x 106 BMA CD34+ x 106 BMC % Yield TNC % Yield MNC % Yield CD34
1386

+/- 383

823

+/-172

270

+/-70

190

+/-79

12.64

+/-7.67

9.02

+/-4.3

60.8

+/-10.6

70.2

+/-21.9

74.6

+/-13.7

 

 

TNC = total nucleated cells                            BMA = bone marrow aspirate

MNC = mononuclear cells                              BMC = bone marrow concentrate

 

 

Table 2:  MEAN VALUES:  N = 3 volunteer donors processed at point of care

Total TNC x 106 BMA Total TNC x 106 BMC Total MNC x 106 BMA Total MNC x 106 BMC CD34+ x 106 BMA CD34+ x 106 BMC % Yield TNC % Yield MNC % Yield CD34
1331

+/- 212

835

+/-88

364

+/-114

237

+/-54

21.3

+/-8

14.1

+/-5.2

67.7

+/-4.2

65.8

+/-11.1

68.6

+/-19.1

 

t-test was performed comparing the two groups.  P = 0.05 significant.

TNC% Yield:   P=0.29.  Not significantly different

MNC% Yield:  P=0.74.  Not significantly different.

CD34+% Yield:  P=0.53.  Not significantly different.

 

We have also compared the number of fibroblast colony-forming units (CFU’s) prepared by the Harvest system with that obtained using a cell separator.  The data is shown below.

Conclusions:

  • Bone marrow derived stem cells can be concentrated at point of care with 70% efficiency within 15 minutes.
  • The system can effectively and reproducibly collect mononuclear cells in concentrations up to 7 times native levels, while maintaining the viability of the cellular components.
  • The product can be red cell reduced for percutaneous intracoronary, or endocardial administration with an additional two minutes of preparation time.