Abstract
Background: There is growing interest in the use of dendritic cells (DCs) for treatment of malignancy and infectious disease. Our goal was to develop a clinical-scale method to prepare autologous DCs for cancer clinical trials. Methods: PBMC were collected from normal donors or cancer patients by automated leukapheresis, purified by counterflow centrifugal elutriation and placed into culture in polystyrene flasks at 1 × 106 cells/mL for 5-7 days at 37°C, with 5% CO2, with IL-4 and GM-CSF. Conditions investigated included media formulation, supplementation with heat-inactivated allogeneic AB serum or autologous plasma and time to harvest (Day 5 or Day 7). DCs were evaluated for morphology, quantitative yield, viability, phenotype and function, including mixed leukocyte response and recall response to tetanus toxoid and influenza virus. Results: DCs with a typical immature phenotype (CD14-negative, CD1a-positive, mannose receptor-positive, CD80-positive, CD83-negative) were generated most consistently in RPMI 1640 supplemented with 10% allogeneic AB serum or 10% autologous plasma. Cell yield was higher at Day 5 than Day 7, without detectable differences in phenotype or function. In pediatric sarcoma patients, autologous DCs had enhanced function compared with monocytes from which they were generated. In this patient group, starting with 8.0± 3.7× 108 fresh or cryopreserved autologous monocytes, DC yield was 2.1±1.0 × 108 cells, or 29% of the starting monocyte number. Discussion: In the optimized clinical-scale method, purified peripheral monocytes are cultured for 5 days in flasks at 1 × 106 cells/mL in RPMI 1640, 10% allogeneic AB serum or autologous plasma, IL-4 and GM-CSF. This method avoids the use of FBS and results in immature DCs suitable for clinical trials.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 19-29 |
| Number of pages | 11 |
| Journal | Cytotherapy |
| Volume | 3 |
| Issue number | 1 |
| DOIs | |
| State | Published - 2001 |
| Externally published | Yes |
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Keywords
- Dendritic cells (DCs)
- Immunotherapy
- Peripheral blood monocytes
ASJC Scopus subject areas
- Immunology
Cite this
Development of a clinical-scale method for generation of dendritic cells from PBMC for use in cancer immunotherapy. / Wong, E. C C; Maher, V. E.; Hines, K.; Lee, J.; Carter, C. S.; Goletz, T.; Kopp, W.; Mackall, C. L.; Berzofsky, J. A.; Read, E. J.
In: Cytotherapy, Vol. 3, No. 1, 2001, p. 19-29.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Development of a clinical-scale method for generation of dendritic cells from PBMC for use in cancer immunotherapy
AU - Wong, E. C C
AU - Maher, V. E.
AU - Hines, K.
AU - Lee, J.
AU - Carter, C. S.
AU - Goletz, T.
AU - Kopp, W.
AU - Mackall, C. L.
AU - Berzofsky, J. A.
AU - Read, E. J.
PY - 2001
Y1 - 2001
N2 - Background: There is growing interest in the use of dendritic cells (DCs) for treatment of malignancy and infectious disease. Our goal was to develop a clinical-scale method to prepare autologous DCs for cancer clinical trials. Methods: PBMC were collected from normal donors or cancer patients by automated leukapheresis, purified by counterflow centrifugal elutriation and placed into culture in polystyrene flasks at 1 × 106 cells/mL for 5-7 days at 37°C, with 5% CO2, with IL-4 and GM-CSF. Conditions investigated included media formulation, supplementation with heat-inactivated allogeneic AB serum or autologous plasma and time to harvest (Day 5 or Day 7). DCs were evaluated for morphology, quantitative yield, viability, phenotype and function, including mixed leukocyte response and recall response to tetanus toxoid and influenza virus. Results: DCs with a typical immature phenotype (CD14-negative, CD1a-positive, mannose receptor-positive, CD80-positive, CD83-negative) were generated most consistently in RPMI 1640 supplemented with 10% allogeneic AB serum or 10% autologous plasma. Cell yield was higher at Day 5 than Day 7, without detectable differences in phenotype or function. In pediatric sarcoma patients, autologous DCs had enhanced function compared with monocytes from which they were generated. In this patient group, starting with 8.0± 3.7× 108 fresh or cryopreserved autologous monocytes, DC yield was 2.1±1.0 × 108 cells, or 29% of the starting monocyte number. Discussion: In the optimized clinical-scale method, purified peripheral monocytes are cultured for 5 days in flasks at 1 × 106 cells/mL in RPMI 1640, 10% allogeneic AB serum or autologous plasma, IL-4 and GM-CSF. This method avoids the use of FBS and results in immature DCs suitable for clinical trials.
AB - Background: There is growing interest in the use of dendritic cells (DCs) for treatment of malignancy and infectious disease. Our goal was to develop a clinical-scale method to prepare autologous DCs for cancer clinical trials. Methods: PBMC were collected from normal donors or cancer patients by automated leukapheresis, purified by counterflow centrifugal elutriation and placed into culture in polystyrene flasks at 1 × 106 cells/mL for 5-7 days at 37°C, with 5% CO2, with IL-4 and GM-CSF. Conditions investigated included media formulation, supplementation with heat-inactivated allogeneic AB serum or autologous plasma and time to harvest (Day 5 or Day 7). DCs were evaluated for morphology, quantitative yield, viability, phenotype and function, including mixed leukocyte response and recall response to tetanus toxoid and influenza virus. Results: DCs with a typical immature phenotype (CD14-negative, CD1a-positive, mannose receptor-positive, CD80-positive, CD83-negative) were generated most consistently in RPMI 1640 supplemented with 10% allogeneic AB serum or 10% autologous plasma. Cell yield was higher at Day 5 than Day 7, without detectable differences in phenotype or function. In pediatric sarcoma patients, autologous DCs had enhanced function compared with monocytes from which they were generated. In this patient group, starting with 8.0± 3.7× 108 fresh or cryopreserved autologous monocytes, DC yield was 2.1±1.0 × 108 cells, or 29% of the starting monocyte number. Discussion: In the optimized clinical-scale method, purified peripheral monocytes are cultured for 5 days in flasks at 1 × 106 cells/mL in RPMI 1640, 10% allogeneic AB serum or autologous plasma, IL-4 and GM-CSF. This method avoids the use of FBS and results in immature DCs suitable for clinical trials.
KW - Dendritic cells (DCs)
KW - Immunotherapy
KW - Peripheral blood monocytes
UR - http://www.scopus.com/inward/record.url?scp=17744371122&partnerID=8YFLogxK
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U2 - 10.1080/146532401753156377
DO - 10.1080/146532401753156377
M3 - Article
VL - 3
SP - 19
EP - 29
JO - Cytotherapy
JF - Cytotherapy
SN - 1465-3249
IS - 1
ER -