Influence of 2 cryopreservation methods to induce CCL-13 from dental pulp cells.
BACKGROUND
Cryopreservation preserves periodontal ligament cells but has a lower success rate with dental pulp cells (DPCs) because it causes inflammation. There are 2 well-known cryopreservation methods that reduce inflammation, slow freezing and rapid freezing, but the effects of the 2 methods on inflammation are not well-established. The purpose of this study was to compare the effects of the 2 different cryopreservation methods on CCL-13 induction from DPCs by using microarrays, real-time polymerase chain reaction (PCR), Western blotting, enzyme-linked immunosorbent assay, and confocal laser scanning microscopy (CLSM).


METHODS
In this study, the concentration of cryoprotectant was fixed, and the methods compared differed with respect to freezing speed. Initially we screened the DPCs of cryopreserved teeth with expression microarrays, and CCL-13 was identified as a differentially expressed gene involved in generalized inflammation. We then compared the expression of CCL-13 after exposing teeth to the 2 cryopreservation methods by using real-time PCR, Western blot, enzyme-linked immunosorbent assay, and CLSM.


RESULTS
Expression of CCL-13 was up-regulated significantly only in the rapid freezing group, except in measurements made by real-time PCR. CLSM analysis also confirmed this up-regulation visually.


CONCLUSIONS
Rapid freezing increased the expression of CCL-13 in DPCs compared with slow freezing. Understanding the inflammatory effect of cryopreservation should help to establish an optimal cryoprofile to minimize inflammation of DPCs and reduce the need for endodontic treatment.
Department of Prosthodontics, School of Dentistry, Kyung Hee University, Seoul, Korea.
Su-Jin
SJ
Ahn
Ji-Hyun
JH
Jang
Ji-Sung
JS
Seo
Kyu Min
KM
Cho
Su-Hee
SH
Jung
Hyeon-Woo
HW
Lee
Eun-Cheol
EC
Kim
Sang Hyuk
SH
Park
D015153:Blotting, Western; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000627:therapeutic use; D003782:Dental Pulp / Q000166:cytology* / Q000378:metabolism; D004121:Dimethyl Sulfoxide / Q000627:therapeutic use; D004797:Enzyme-Linked Immunosorbent Assay; D005615:Freezing; D006801:Humans; D046228:Microarray Analysis; D018613:Microscopy, Confocal; D018945:Monocyte Chemoattractant Proteins / Q000032:analysis / Q000096:biosynthesis*; D009584:Nitrogen; D060888:Real-Time Polymerase Chain Reaction; D013997:Time Factors; D015854:Up-Regulation
C099677:CCL13 protein, human; D003451:Cryoprotective Agents; D018945:Monocyte Chemoattractant Proteins; D009584:Nitrogen; D004121:Dimethyl Sulfoxide
Autotransplantation; CCL-13; cryopreservation; dental pulp cell; inflammation