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Chamber probe position effects on controlled rate freezing
S40
Poster Abstract Presentations
94% with function maintained compared to pre-freeze. The results of the study demonstrate that cryopreservation can be a valuable option for maximizing the stability and quality of the starting material for cell therapies 109 CHAMBER PROBE POSITION EFFECTS ON CONTROLLED RATE FREEZING E.J. Gilles, D. Gastineau & E. Jacob Human Cellular Therapy, Mayo Clinic, Rochester, Minnesota, United States Background & Aim: Background: Controlled Rate Freezers (CRF) enable long-term cryopreservation of cellular therapy products. Freezing curves record that cryopreservation temperatures are following optimized protocols. Two abnormal freezing curves were investigated. Methods, Results & Conclusion Method: Retrospective case reviews. Results: In the first case, the standard freezing protocol was followed with minor cyclic oscillations. Product integrity was not compromised. The CRF has a wire chamber temperature probe that makes a 90 degree bend and descends through a six inch metal straw which prevents liquid nitrogen vapor from directly contacting the probe. The insulated probe is uncoated for 3 mm on the distal protruding end allowing for accurate measurements. The straw and probe end are not visible in the CRF due to a metal plate. Upon inspection by the vendor after first abnormal curve, the chamber probe was not protruding far enough out of the straw. Repositioning counteracted the observed oscillations. Several months later, a second abnormal freezing curve was observed. Ten minutes into the run, it was noted that the chamber temperature was having drastic, wide-ranging swings. The sample temperature did not show the irregular swings, but indicated warming. Upon inspection, the uncoated tip of the probe was making contact with the metal straw. Both types of oscillation were reproducible by varying the position of the chamber probe. The exposed temperature probe allows the CRF to measure the temperature of the chamber and inject liquid nitrogen vapor into the system to allow for cooling. When the probe is not in the proper position it is either insulated by the straw (probe within the straw) or the probe is grounded and produces erroneous temperature swings (when the probe touches the metal straw). In the latter case, erroneously cold chamber temperatures occurred, producing large-scale temperature oscillations on the chamber probe which caused the CRF to respond with rapid warming. Product integrity was compromised by the second, more severe event. The vendor was called to discuss the findings and they reported other clients had similar experiences with the grounding of the probe. Currently, we ensure the chamber probe is pulled at least 1 cm through the straw and bent at a 90 degree angle.
An illustration of when the end of the probe is pulled into the metal tube, but not making contact with metal. The chamber curve shows dramatic, uniform oscillations and the sample curve shows smaller oscillations.
An illustration of when the probe makes contact with the metal tube. The grounding causes large, rapid temperature measurement deviations. Because the freezer is incorrectly detecting the chamber temperature to be low (in relation to the profile), the freezer responds by warming up, as is evident when looking at the sample curve.
Conclusion: Probe position is essential for proper function of the CRF which may affect cryopreservation of products. Any oscillations in the freezing curve necessitate evaluation of the chamber probe position. 111 EVALUATING AUTOMATED BUFFER EXCHANGE PROTOCOLS USING ROTEATM COUNTERFLOW CENTRIFUGE A. Li12, S. Wilson3, I. Fitzpatrick3, S. Chan1,2, M. Barabadi1,2, G. Kusuma1,2, D. James3 & R. Lim1,2,4 1 The Ritchie Centre, Hudson Institute of Medical Research, Clayton, Victoria, Australia, 2Department of Obstetrics and Gynaecology, Monash University, Clayton, Victoria, Australia, 3Scinogy Pty Ltd, Melbourne, Victoria, Australia, 4Australian Regenerative Medicine Institute, Clayton, Victoria, Australia Background & Aim: Buffer exchange is an essential process in cell therapy manufacturing where the introduction of automation can reduce costs and enable commercial scale manufacture. RoteaTM (Scinogy), is a benchtop counterflow centrifuge specifically designed for cell therapy processing, that allows closed automated buffer exchange for small to medium size batches. To determine the capability of RoteaTM , we compared the automated protocols to manual centrifugation for buffer exchange using cultured Jurkat cells and mesenchymal stromal cells (MSCs). Methods, Results & Conclusion Methods: The details of starting material and wash buffer for various cell types are listed in Table 1. In both automated protocols, wash buffer and cells were loaded into the cell transfer bags and connected onto the single-use processing kit. The automated process starts with loading the cells into the cell chamber, where they were then washed with 40 ml of the buffer before being concentrated to a final volume of 10 ml. The automated processes were compared to two cycles of centrifugation and resuspension. The recovered cells were also plated for MTS assays to measure cell proliferation over 48 hours. Results: Automated buffer exchange has a shorter processing time compared to the manual process. Cell viabilities were generally improved after buffer exchange and cell recoveries were close to 100% (Table 1). Cell proliferation rates were similar between manual and automated processes. Conclusions: The closed and automated counterflow centrifuge is ideally suited to perform buffer exchange for various cell types with high cell recoveries and shorter processing times. Table 1.
Poster Abstract Presentations
94% with function maintained compared to pre-freeze. The results of the study demonstrate that cryopreservation can be a valuable option for maximizing the stability and quality of the starting material for cell therapies 109 CHAMBER PROBE POSITION EFFECTS ON CONTROLLED RATE FREEZING E.J. Gilles, D. Gastineau & E. Jacob Human Cellular Therapy, Mayo Clinic, Rochester, Minnesota, United States Background & Aim: Background: Controlled Rate Freezers (CRF) enable long-term cryopreservation of cellular therapy products. Freezing curves record that cryopreservation temperatures are following optimized protocols. Two abnormal freezing curves were investigated. Methods, Results & Conclusion Method: Retrospective case reviews. Results: In the first case, the standard freezing protocol was followed with minor cyclic oscillations. Product integrity was not compromised. The CRF has a wire chamber temperature probe that makes a 90 degree bend and descends through a six inch metal straw which prevents liquid nitrogen vapor from directly contacting the probe. The insulated probe is uncoated for 3 mm on the distal protruding end allowing for accurate measurements. The straw and probe end are not visible in the CRF due to a metal plate. Upon inspection by the vendor after first abnormal curve, the chamber probe was not protruding far enough out of the straw. Repositioning counteracted the observed oscillations. Several months later, a second abnormal freezing curve was observed. Ten minutes into the run, it was noted that the chamber temperature was having drastic, wide-ranging swings. The sample temperature did not show the irregular swings, but indicated warming. Upon inspection, the uncoated tip of the probe was making contact with the metal straw. Both types of oscillation were reproducible by varying the position of the chamber probe. The exposed temperature probe allows the CRF to measure the temperature of the chamber and inject liquid nitrogen vapor into the system to allow for cooling. When the probe is not in the proper position it is either insulated by the straw (probe within the straw) or the probe is grounded and produces erroneous temperature swings (when the probe touches the metal straw). In the latter case, erroneously cold chamber temperatures occurred, producing large-scale temperature oscillations on the chamber probe which caused the CRF to respond with rapid warming. Product integrity was compromised by the second, more severe event. The vendor was called to discuss the findings and they reported other clients had similar experiences with the grounding of the probe. Currently, we ensure the chamber probe is pulled at least 1 cm through the straw and bent at a 90 degree angle.
An illustration of when the end of the probe is pulled into the metal tube, but not making contact with metal. The chamber curve shows dramatic, uniform oscillations and the sample curve shows smaller oscillations.
An illustration of when the probe makes contact with the metal tube. The grounding causes large, rapid temperature measurement deviations. Because the freezer is incorrectly detecting the chamber temperature to be low (in relation to the profile), the freezer responds by warming up, as is evident when looking at the sample curve.
Conclusion: Probe position is essential for proper function of the CRF which may affect cryopreservation of products. Any oscillations in the freezing curve necessitate evaluation of the chamber probe position. 111 EVALUATING AUTOMATED BUFFER EXCHANGE PROTOCOLS USING ROTEATM COUNTERFLOW CENTRIFUGE A. Li12, S. Wilson3, I. Fitzpatrick3, S. Chan1,2, M. Barabadi1,2, G. Kusuma1,2, D. James3 & R. Lim1,2,4 1 The Ritchie Centre, Hudson Institute of Medical Research, Clayton, Victoria, Australia, 2Department of Obstetrics and Gynaecology, Monash University, Clayton, Victoria, Australia, 3Scinogy Pty Ltd, Melbourne, Victoria, Australia, 4Australian Regenerative Medicine Institute, Clayton, Victoria, Australia Background & Aim: Buffer exchange is an essential process in cell therapy manufacturing where the introduction of automation can reduce costs and enable commercial scale manufacture. RoteaTM (Scinogy), is a benchtop counterflow centrifuge specifically designed for cell therapy processing, that allows closed automated buffer exchange for small to medium size batches. To determine the capability of RoteaTM , we compared the automated protocols to manual centrifugation for buffer exchange using cultured Jurkat cells and mesenchymal stromal cells (MSCs). Methods, Results & Conclusion Methods: The details of starting material and wash buffer for various cell types are listed in Table 1. In both automated protocols, wash buffer and cells were loaded into the cell transfer bags and connected onto the single-use processing kit. The automated process starts with loading the cells into the cell chamber, where they were then washed with 40 ml of the buffer before being concentrated to a final volume of 10 ml. The automated processes were compared to two cycles of centrifugation and resuspension. The recovered cells were also plated for MTS assays to measure cell proliferation over 48 hours. Results: Automated buffer exchange has a shorter processing time compared to the manual process. Cell viabilities were generally improved after buffer exchange and cell recoveries were close to 100% (Table 1). Cell proliferation rates were similar between manual and automated processes. Conclusions: The closed and automated counterflow centrifuge is ideally suited to perform buffer exchange for various cell types with high cell recoveries and shorter processing times. Table 1.