An improved method for processing chondroprogenitor pellets following chondrogenic differentiation for histology and immunohistochemical staining using agarose
Soosai Manickam Amirtham , Upasana Kachroo , Deepak Vinod Francis , Kawin Padmaja , Elizabeth Vinod
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引用次数: 0
Abstract
Purpose
In-vitro models of cartilage regeneration based on pellet cultures have been widely used to evaluate chondrogenic potential of the cell of interest and predict probable in-vivo behavior. However, pellet processing is a major challenge during handling (due to small size and possible damage to structural contour following sectioning and staining). The present study aimed to utilize human articular cartilage derived chondroprogenitors to assess if agarose-encapsulation of pellets prior to paraffin processing enable easier handling without affecting tissue morphology, glycosaminoglycan staining and immunohistochemical analysis of Collagen type II protein.
Methods
Passage 3 chondroprogenitors (n = 3) were evaluated for MSC markers using flow cytometry and subjected to chondrogenic differentiation as pellets cultures. Post-differentiation, the pellets were subjected to either: a) paraffin embedding, b) agarose encapsulation followed by paraffin embedding or c) agarose encapsulation followed by cryosectioning. All sections were subjected to histological staining for glycosaminoglycan uptake: Alcian blue, Safranin O (Bern score) and Toluidine blue with immunohistochemical processing for collagen type II protein deposition.
Results
With respect to staining and structural integrity, comparable uptake was seen in both paraffin sections and agarose embedded sections while the latter exhibited notably uniform pellets with distinct marginal demarcation. Although plain paraffin and agarose encapsulated sections demonstrated equivalent staining as represented by comparable Bern scores, glycosaminoglycan uptake, and Collagen type II deposition, cryosections exhibited significantly poor staining properties.
Conclusion
Agarose encapsulation of differentiated pellets prior to routine paraffin embedding, eases handling difficulties whilst maintaining structural integrity with optimal staining outcomes.
期刊介绍:
Journal of Arthroscopy and Joint Surgery (JAJS) is committed to bring forth scientific manuscripts in the form of original research articles, current concept reviews, meta-analyses, case reports and letters to the editor. The focus of the Journal is to present wide-ranging, multi-disciplinary perspectives on the problems of the joints that are amenable with Arthroscopy and Arthroplasty. Though Arthroscopy and Arthroplasty entail surgical procedures, the Journal shall not restrict itself to these purely surgical procedures and will also encompass pharmacological, rehabilitative and physical measures that can prevent or postpone the execution of a surgical procedure. The Journal will also publish scientific research related to tissues other than joints that would ultimately have an effect on the joint function.