Modification of the diphenylamine assay for cell quantification in three-dimensional biodegradable polymeric scaffolds.
As three-dimensional (3D) cell culture systems gain popularity in biomedical research, reliable assays for cell proliferation within 3D matrices become more important. Although many cell quantification techniques have been established for cells cultured on nondegradable plastic culture dishes and cells suspended in media, it is becoming increasingly clear that cell quantification after prolonged culture in 3D polymeric scaffolds imposes unique challenges because the added presence of polymeric materials may contribute to background signal via various mechanisms including autofluorescence, diffusion gradients, and sequestering effects. Thus, additional steps are required to ensure complete isolation of cells from the 3D scaffold. The diphenylamine assay isolates cellular DNA, degrades the polymeric matrix materials, and reacts with the DNA to yield a colorimetric response. Thus, we report here a practical modification of the diphenylamine assay and show that the assay quantifies cells in 3D polyester scaffolds reliably and reproducibly as long as the necessary amount of the acidic working reagent is present. Our study also demonstrates that the sensitivity of the assay can be optimized by controlling the dimensions of the sampling volume. Overall, the DPA assay offers an attractive solution for challenges associated with 3D cell quantification.
Pham
Edward A
EA
Department of Bioengineering, University of California, Los Angeles, California 90095, USA.
Ho
Won Jin
WJ
Kamei
Daniel T
DT
Wu
Benjamin M
BM
D020341:Absorbable Implants; D001672:Biocompatible Materials; D002452:Cell Count / Q000379:methods*; D002460:Cell Line; D004247:DNA / Q000096:biosynthesis / Q000302:isolation & purification; D004159:Diphenylamine; D005470:Fluorometry; D006801:Humans; D007202:Indicators and Reagents; D019344:Lactic Acid; D011091:Polyesters; D011100:Polyglycolic Acid; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011108:Polymers; D011474:Prosthesis Design; D012015:Reference Standards; D054457:Tissue Scaffolds
D001672:Biocompatible Materials; D007202:Indicators and Reagents; D011091:Polyesters; D011108:Polymers; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; C016240:polycaprolactone; D011100:Polyglycolic Acid; D019344:Lactic Acid; D004247:DNA; D004159:Diphenylamine