Journal: BioResearch Open Access

Article Title: Effect of Cryopreservation on Canine and Human Activated Nucleus Pulposus Cells: A Feasibility Study for Cell Therapy of the Intervertebral Disc

doi: 10.1089/biores.2013.0023

Figure Lengend Snippet: (A) The harvested nucleus pulposus (NP) tissue was separated into three groups (Group A, noncryopreservation group; Group B, cryopreservation cell group; Group C, cryopreservation tissue group). The NP tissue of Groups A and B was enzymatically digested immediately. In Group A, NP cells were cocultured in accordance with the protocols used in a pilot clinical study currently being conducted by our research group as shown. In Group B, cells were cryopreserved for 2 weeks and were cocultured after thawing. The NP tissue of Group C was sliced finely and cryopreserved for 2 weeks in thin tissue sections. After the samples were thawed, they were treated with enzymes and cocultured in accordance with the culture protocols used in Groups A and B. (B) NP cells or tissue or the mononuclear cells were preserved in 70% Dulbecco's modified Eagle's medium (DMEM)/F12, 20% fetal bovine serum (FBS), and 10% dimethylsulfoxide in a cryotube. Next, the samples were cryopreserved in stages to −80°C using a controlled-rate cryopreservation device. The next day, the samples were stored in a liquid nitrogen container at −196°C.

Article Snippet: Next, the samples were cryopreserved in stages to −80°C by using a controlled-rate cryopreservation device (Bicell, Nihon Freezer, Tokyo, Japan).

Techniques: Modification

Journal: BioResearch Open Access

Article Title: Effect of Cryopreservation on Canine and Human Activated Nucleus Pulposus Cells: A Feasibility Study for Cell Therapy of the Intervertebral Disc

doi: 10.1089/biores.2013.0023

Figure Lengend Snippet: The mRNA expression of activated NP cells of the noncryopreservation group and cryopreservation group of human cells were compared using reverse-transcription polymerase chain reaction (RT-PCR). After cells were cultured for 7 days, total RNA was isolated from the activated NP cells using the Total RNA Isolation System, reverse transcribed to cDNA using a High Capacity RNA-to-cDNA Kit, and amplified by PCR using specific primers for each gene. The RT-PCR results for all the evaluated genes were the same for all three groups, and no effects of cryopreservation were observed.

Article Snippet: Next, the samples were cryopreserved in stages to −80°C by using a controlled-rate cryopreservation device (Bicell, Nihon Freezer, Tokyo, Japan).

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Isolation, Amplification

Journal: BioResearch Open Access

Article Title: Effect of Cryopreservation on Canine and Human Activated Nucleus Pulposus Cells: A Feasibility Study for Cell Therapy of the Intervertebral Disc

doi: 10.1089/biores.2013.0023

Figure Lengend Snippet: The activated human NP cells from the three groups were checked for presence of chromosomal abnormalities. There was no evidence of chromosomal abnormalities in any culture. There were no negative effects on the cell cycle of activated NP cells after cryopreservation and thawing.

Article Snippet: Next, the samples were cryopreserved in stages to −80°C by using a controlled-rate cryopreservation device (Bicell, Nihon Freezer, Tokyo, Japan).

Techniques:

Journal: BioResearch Open Access

Article Title: Effect of Cryopreservation on Canine and Human Activated Nucleus Pulposus Cells: A Feasibility Study for Cell Therapy of the Intervertebral Disc

doi: 10.1089/biores.2013.0023

Figure Lengend Snippet: Activated human NP cells from the three groups were suspended in PBS and subcutaneously injected into NOD-SCID mice. After 8 weeks, no noticeable tuberculate appearance under the skin of the mice was observed. Membranes of the transplanted portions were harvested and stained with hematoxylin–eosin and safranin O. The nuclei were regularly aligned and the cell size was uniform. There was no evidence indicating tumorigenesis of activated NP cells (Group A, noncryopreservation group; Group B, cell cryopreservation group; Group C, tissue cryopreservation group). Magnifications 40×.

Article Snippet: Next, the samples were cryopreserved in stages to −80°C by using a controlled-rate cryopreservation device (Bicell, Nihon Freezer, Tokyo, Japan).

Techniques: Injection, Staining

Journal: International Journal of Molecular Sciences

Article Title: Investigation of the Mitigation of DMSO-Induced Cytotoxicity by Hyaluronic Acid following Cryopreservation of Human Nucleus Pulposus Cells

doi: 10.3390/ijms241512289

Figure Lengend Snippet: Simulation of NPC transplantation in degenerating IVD in vitro. Cryopreservation solution was not removed from thawed NPCs and mixed in equal volumes with 1 mL each of A-EDTA (Group E) or ARTZ ® (1% HA solution) (Group H); after 3–5 h of incubation, DMSO was removed as if diffusion metabolism occurred in IVD (DMSO concentration less than 1%) and cultured for 5 days.

Article Snippet: After collecting the cultured cells, they were aliquoted into a cryotube at 3.0 × 10 5 cells in 1 mL of CryoStor ® CS10 (CS10) (STEMCELL Technologies, Vancouver, BC, Canada), which contains 10% DMSO, and the samples were cryopreserved in stages to −80 °C using a controlled-rate cryopreservation device (Bicell, Nihon Freezer, Tokyo, Japan).

Techniques: Transplantation Assay, In Vitro, Incubation, Diffusion-based Assay, Concentration Assay, Cell Culture