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Reversal of senescence-associated beta-galactosidase expression during in vitro 3D TE

Reversal of senescence-associated beta-galactosidase expression during in vitro three-dimensional tissue-engineering of human chondrocytes in a polymer scaffold


Regenerative medicine applications require cells that are not inflicted with senescence after in vitro culture for an optimal in vivo outcome. Methods to overcome replicative senescence include genomic modifications which have their own disadvantages. They have evaluated a three-dimensional (3D) thermo-reversible gelation polymer (TGP) matrix environment for its capabilities to reverse cellular senescence. The expression of senescence-associated beta-galactosidase (SA-βgal) by human chondrocytes from osteoarthritis-affected cartilage tissue, grown in a conventional two-dimensional (2D) monolayer culture versus in 3D-TGP were compared. In 2D, the cells de-differentiated into fibroblasts, expressed higher SA-βgal and started degenerating at 25 days. SA-βgal levels decreased when the chondrocytes were transferred from the 2D to the 3D-TGP culture, with cells exhibiting a tissue-like growth until 42–45 days. Other senescence associated markers such as p16INK4a and p21 were also expressed only in 2D cultured cells but not in 3D-TGP tissue engineered cartilage. This is a first-of-its-kind report of a chemically synthesized and reproducible in vitro environment yielding an advantageous reversal of aging of human chondrocytes without any genomic modifications. The method is worth consideration as an optimal method for growing cells for regenerative medicine applications.


Illustration of the study groups and time-points of evaluation of SA-βgal, p16INK4a and p21 in two-dimensional (2D) and three-dimensional (3D) thermo-reversible gelation polymer (3D-TGP) cultures.


Cell and tissue engineering-based regenerative therapies warrant good-quality cells and tissues for optimal clinical outcomes. Cellular senescence is a multifaceted process that arrests cell proliferation. The term was first mentioned in the landmark paper by Leonard Hayflick, who reported that in vitro cultured primary human fibroblasts have a restricted lifespan, which is approximately 50 cell divisions, known as “Hayflick’s limit”.


(A,B) In vitro culture images: (A) Chondrocytes in two-dimensional (2D) culture de-differentiating into fibroblast-like cells; (B) In vitro cultured chondrocytes growing in a tissue-like manner in three-dimensional (3D) thermo-reversible gelation polymer (3D-TGP) culture (the red arrow indicates the tissue; the black arrow indicates the cells migrating out into the 3D environment into the tissue); (C,D) H-and-E staining images: (C) Chondrocytes in 2D observed as individual cells. (D) 3D-TGP tissue-engineered chondrocytes exhibiting continuous tissue morphology with hyaline phenotype; (E,F) Safranin O/Fast Green staining images: (E) Chondrocytes in 2D observed as individual cells. (F) 3D-TGP tissue-engineered chondrocytes exhibiting continuous tissue morphology; (G,H) Toluidine blue images: (E) Chondrocytes in 2D observed as individual cells. (F) 3D-TGP tissue-engineered chondrocytes exhibiting continuous tissue morphology (All scale bars = 100 μm).


A tissue’s ability to regenerate decreases when a significant proportion of proliferating cells undergoes cellular senescence. The number of senescent cells increases with age in multiple types of tissues. Cellular senescence is triggered in response to a variety of stressors, including telomere shortening, oxidative stress, DNA damage, and oncogene activation. Telomere shortening is the major cause underlying replicative senescence. While most human somatic cell types express little or no telomerase activity, leading to telomere loss, and proliferating normal stem cells though express regulated telomerase, the expression level is insufficient to maintain telomeres, and gradual telomere erosion occurs. Progressive telomere shortening leads to in vitro replicative senescence.


Relative expression of p16 INK4a and p21 only in 2D cultured chondrocytes and not in 3D-TGP indicating presence of senescent cells in 2D cultures but not in 3D-TGP.


Regarding age-related diseases like osteoarthritis (OA), chondrocytes primarily are thought to play a major role in OA induction as they become senescent due to progressive telomere shortening with age. Senescent chondrocytes are absent from normal cartilage and are present near osteoarthritic lesions. When such senescent cells were transplanted into the knee joint of wild type mice, an OA-like state was induced, thus showing that senescence of chondrocytes is a major factor driving OA. When chondrocytes are cultured in vitro, especially in monolayer, they easily lose their native phenotype, de-differentiate and express various senescence- and dedifferentiation-related genes. Replicative senescence in vitro has been observed after 30–40 passages during in vitro culture of chondrocytes, which then exhibit features of the senescent phenotype, including enlarged flattened cells in culture and the expression of SA-βgal. The cellular senescence of in vitro cultured cells is usually overcome by inducing telomerase activity or initiating recombination-mediated alternative lengthening of telomeres (ALT) pathway(s) or genomic modifications such as reprogramming using specific transcription factors, all of which carry a risk of oncogenesis.


Mean fluorescence intensity (ΔG MFI) of the expression of SA-βgal evaluated by flow cytometry in 2D compared to 3D-TGP at different durations of culture with 2D-cultured chondrocytes (evaluation I and II) showing higher levels of SA-βgal as culture, while the 3D-cultured cells (evaluation III and IV) showed very low levels of SA-βgal throughout the culture period. (A) Sample 1; (B) Sample 2 (FSC(High)); (C) Sample 2 (FSC(Low)); (D) Sample 3 (FSC(High)); (E) Sample 3 (FSC(Low)). I, II, III and IV denote the Senescence-associated beta-galactosidase (SA-βgal) evaluation time-points


An in vitro culture method which does not involve any such genomic modifications would be ideal for use in regenerative therapies. The capabilities of a three-dimensional (3D) thermo-reversible gelation polymer (TGP) to maintain the native phenotype for a longer time in vitro have been reported for several cell types such as corneal endothelial precursor cells, corneal limbal stem cells, mesenchymal stem cells, buccal epithelial cellsand chondrocytes.


Gating in the flow cytometric analysis of osteoarthritic chondrocytes grown in 2D and 3D-TGP with the SA-βgal expression in two heterogeneous populations sorted by flow cytometry (FSChigh versus FSClow) with the 2D-grown cells showing higher SA-βgal expression than 3D-TGP cultured cells on both day 26 and day 42 of culture (A) Day 26; (B) Day 42. II, III and IV denote the Senescence-associated beta-galactosidase (SA-βgal) evaluation time-points.


This 3D-TGP can maintain the native hyaline phenotype of knee-cartilage-derived chondrocytes from bovine, rabbit, and human sources for 16–18 weeks, both in vitro and in vivo. In vitro 3D-TGP-tissue-engineered cartilage tissue expressed pluripotency-related markers in a lectin micro-array, higher miRNA21 and 140 expression indicative of healthy cartilage phenotyp and mesenchymal-chondroprogenitor markers. They sought to examine the expression of senescence-associated beta-galactosidase (SA-βgal) in human chondrocytes derived from elderly donors affected by OA, cultured in 2D- followed by 3D-TGP.


  1. Katoh, S., Fujimaru, A., Iwasaki, M. et al. Reversal of senescence-associated beta-galactosidase expression during in vitro three-dimensional tissue-engineering of human chondrocytes in a polymer scaffold. Sci Rep11, 14059 (2021). https://doi.org/10.1038/s41598-021-93607-9