Inclusion of macromolecular crowders in three-dimensional human gingival fibroblast cultures

doi:10.21203/rs.3.pex-2171/v1

Method Article

Inclusion of macromolecular crowders in three-dimensional human gingival fibroblast cultures

https://doi.org/10.21203/rs.3.pex-2171/v1

This work is licensed under a CC BY 4.0 License

This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.

Version 1

posted

You are reading this older protocol version

Read the latest protocol version →

Fibroblast-derived extracellular matrix (ECM) niche regulates cell phenotype and behavior relevant to development, tissue homeostasis, wound healing, and pathological conditions. Improved understanding of cell-ECM interactions requires culture models that mimic the extracellular microenvironment found in vivo. Unlike in vivo, cultured cells reside in aqueous media containing a low concentration of macromolecules. Inclusion of inert macromolecular crowders (MMCs) allows to better mimic in vivo-like conditions in vitro in a phenomenon known as macromolecular crowding (MMC). We present here a standardized protocol to utilize MMCs (Ficoll 70/400) to generate human gingival fibroblast cultures that can be propagated at least for 21 days. The resulting cultures are composed of cells embedded in their own three-dimensional ECM and display gene expression and ECM composition that are distinct from that observed under non-crowded conditions.

Biological techniques

Cell culture

fibroblast

macromolecular crowding

extracellular matrix

Fibroblasts are connective tissue cells that synthesize, assemble, and organize the extracellular matrix (ECM) niche they are situated in. In turn, the ECM regulates the phenotype and function of the resident fibroblasts, in a phenomenon known as dynamic reciprocity¹. The ECM niche is tissue specific and also modulates functions of other cells in the tissue or organ. Understanding these cell-ECM interactions may help to unravel the processes involved in tissue development, homeostasis, pathophysiology, and regeneration. Retaining in vivo-like molecular composition, organization, topography, and mechanical properties of the ECM are imperative in generating culture models that are biologically relevant to in vivo connective tissues. For instance, studies have shown that three-dimensional (3D) fibroblast cultures or matrices (CDMs) mimic some aspects of connective tissues found in vivo, including accumulation of fibrillar proteins (such as fibronectin and collagen I), matricellular proteins (such as tenascin C), proteoglycans and glycosaminoglycans (such as small leucine-rich proteoglycans and hyaluronan, respectively), and growth factors^2,3. Characterization of 3D CDMs from dermal fibroblasts has also shown that ECM topography, elastic modulus, and tensile strength is similar to in vivo dermal connective tissue^2,4.

Thus, 3D cell culture and CDMs may provide a biologically relevant model that could be a valuable tool to study cell-ECM interactions and to be used for regenerative therapy. However, the above findings are based on standard cell culture conditions, which do not fully recapitulate the cellular microenvironment found in vivo. Intracellular and extracellular spaces in vivo are crowded with macromolecules, including nucleic acids, complex sugars, and proteins, in a phenomenon known as macromolecular crowding (MMC)⁵. For example, bone marrow mesenchymal stem cell extracellular environment has about 5 times the concentration of macromolecules compared to cell culture media^6–8. Therefore, the inclusion of synthetic macromolecular crowders (MMCs) into culture media have been proposed to compensate for this deficiency and to further optimize the cultures^5–8. MMCs promote protein folding to a more compact conformation (that may be more stable or enzymatically active) and favors interactions between macromolecules to reduce the occupied (excluded) volume^5,6,9. Crowded environments also reduce diffusion rates of secreted molecules up to 10-fold and concentrate them in the extracellular space^5,6,10,11.

One such macromolecular crowder is Ficoll. Ficoll is a neutral, highly branched, hydrophilic polysaccharide formed by the reaction of sucrose and epichlorohydrin, and dissolves well in aqueous solutions¹². Unlike other crowding agents that are charged, Ficoll does not easily bind to biological molecules allowing it to occupy space without “scavenging” these molecules from the solution. It is commonly synthesized in molecular weights of 70 kDa (Ficoll 70) and 400 kDa (Ficoll 400) with hydrodynamic radii of 40Å and 80Å, respectively. Use of combination of the two different sized crowders increases crowding efficiency compared to a single sized crowder⁷. Use of Ficoll 70 and 400 at 37.5 mg/mL and 25 mg/mL, respectively, results in an estimated fractional volume occupancy (Ψ) of 17% in medium of cultured human fibroblasts or MSCs^7,13, and influences composition and organization of the fibroblast ECM niche in a tissue-type specific manner^7,8,13–19. We have recently adapted the use of MMCs (Ficoll 70/400) to cultures of human gingival fibroblasts²⁰. The following describes the detailed methods to generate these cultures.

Dulbecco’s Modified Eagle’s Medium (DMEM) (CAT#31600-083; Gibco Life Technologies, Grand Island, NY, USA), fetal bovine serum (FBS) (CAT#12484-028; Gibco Life Technologies), antibiotic/antimycotic (CAT#15240-062; Gibco Life Technologies), L-ascorbic acid sodium salt (CAT#A-4034; Millipore Sigma, Burlington, MA, USA), Ficoll 70 (F2878; Millipore Sigma), Ficoll 400 (F4375; Millipore Sigma), HCl (CAT#258148; Sigma Aldrich).

Biosafety cabinet, cell culture incubator, magnetic stirrer, pH meter, beakers, 6-well (9.6 cm²) cell culture plates (CAT#353046; Corning, Corning, NY, USA), bottle top filter (CAT# 596-3320, Thermo Fisher Scientific, Waltham, MA, USA).

Protocol to Prepare DMEM Supplemented with Ficoll 70/400

1. Obtain your desired volume of DMEM and allow it to reach room temperature.

2. Transfer out approximately 10% of the volume and leave it aside in a clean beaker.

3. Transfer the rest of the DMEM to another clean beaker that has volume markings.

4. Place the beaker on a magnetic stirrer and place a clean magnetic mixing bar into the beaker.

5. Weigh out Ficoll 70 and Ficoll 400 such that the final concentration will be 37.5 mg/mL and 25 mg/mL, respectively, for the intended final volume of DMEM to be made.

6. Begin stirring the DMEM at a medium speed (avoid speeds that generate foam).

7. Slowly add Ficoll to the solution. Note that if you add powder too quickly, Ficoll will form clumps that are difficult to completely dissolve.

8. Adding Ficoll to the medium increases the total volume slightly. Therefore, once all Ficoll has been dissolved, turn off the stirrer and note the volume of the medium. Add the required volume of DMEM from the standalone beaker to reach the intended desired final volume.

9. Begin stirring again and place a pH meter in the medium. The addition of Ficoll raises the pH of the solution. Therefore, use a strong acid, such as 12N HCl, to limit the amount of acidic solution needed to adjust the pH to 7.2.

10. Transfer the container to a biosafety cabinet.

11. Assemble a rapid flow 0.2 μm bottle top filter, this will allow for the media to be filtered and collected in a sterile bottle.

12. Add FBS and antibiotic/antimycotic to the media as desired.

13. Medium can be stored at least up to 4 weeks at +4°C.

14. Just before adding medium to the cells, add L-ascorbic acid sodium salt to a final concentration of 50 µg/mL. Note, L-ascorbic acid sodium salt is light sensitive and unstable in aqueous solution and in ambient and higher temperatures. Its stability can be increased by preparing a concentrated stock solution, storing it frozen or at 4°C, and by protection from light^21,22. Therefore, add the ascorbic acid from stock only to an aliquot of media you intend to use on a given day. To prepare the stock, dissolve L-ascorbic acid sodium salt at 50 mg/mL in ultrapure water. Add 1 µL of stock for every 1mL of media to be used. Stock should be aliquoted as appropriate and frozen at -20°C for long term storage. Use a thawed stock aliquot of ascorbic acid within 1-2 weeks and ensure it is stored at 4°C and protected from light.

Cell culture

To generate the cultures (Figure 1), human gingival fibroblasts (seeding density 25,000 cells per cm²) are cultured for up to 21 days in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS), 1% antibiotic/antimycotic, 50 µg/mL L-ascorbic acid sodium salt (to support collagen deposition) and 37.5 mg/mL of Ficoll 70 and 25 mg/mL of Ficoll 400 at 37°C and 5% CO₂on 6-well (9.6 cm²) cell culture plates. For initial cell adhesion, cells are seeded in the culture wells in the above growth medium (3 mL) that does not contain Ficoll 70/400 and ascorbic acid for 24 hours. Medium is then switched to Ficoll 70/400 and ascorbic acid containing medium and cells are cultured for the desired time. Due to limited stability of ascorbic acid in aqueous solution at 37°C²¹ and high cell density in the 3D cultures, medium is changed every other day.

1. Muncie, J. M. & Weaver, V. M. The Physical and Biochemical Properties of the Extracellular Matrix Regulate Cell Fate. in Current Topics in Developmental Biology vol. 130 1–37 (Elsevier Inc., 2018).

2. Kutys, M. L., Doyle, A. D. & Yamada, K. M. Regulation of cell adhesion and migration by cell-derived matrices. Exp. Cell Res. 319, 2434–2439 (2013).

3. Soucy, P. A. & Romer, L. H. Endothelial cell adhesion, signaling, and morphogenesis in fibroblast-derived matrix. Matrix Biol. 28, 273–283 (2009).

4. Cukierman, E., Pankov, R., Stevens, D. R. & Yamada, K. M. Taking Cell-Matrix Adhesions to the Third Dimension. Science. 294, 1708–1712 (2001).

5. Ellis, J. & Minton, A. P. Join the crowd. Nature 425, 27–28 (2003).

6. Ellis, R. J. Macromolecular crowding: obvious but underappreciated. Trends Biochem. Sci. 26, 597–604 (2001).

7. Chen, C., Loe, F., Blocki, A., Peng, Y. & Raghunath, M. Applying macromolecular crowding to enhance extracellular matrix deposition and its remodeling in vitro for tissue engineering and cell-based therapies. Adv. Drug Deliv. Rev. 63, 277–290 (2011).

8. Satyam, A., Kumar, P., Cigognini, D., Pandit, A. & Zeugolis, D. I. Low, but not too low, oxygen tension and macromolecular crowding accelerate extracellular matrix deposition in human dermal fibroblast culture. Acta Biomater. 44, 221–231 (2016).

9. Kuznetsova, I. M., Turoverov, K. K. & Uversky, V. N. What Macromolecular Crowding Dan Do to a Protein. Int. J. Mol. Sci. 15, 23090–23140 (2014).

10. Verkman A. S. Solute and macromolecule diffusion in cellular aqueous compartments. Trends Biochem. Sci. 27, 27–33 (2002).

11. Ellis, J. Macromolecular crowding: an important but neglected aspect of the intracellular environment. Curr. Opin. Struct. Biol. 11, 114–119 (2001).

12. Fissell, W. H. et al. Ficoll is not a rigid sphere. Am. J. Physiol. - Ren. Physiol. 293, 1209–1213 (2007).

13. Ang, X. M. et al. Macromolecular Crowding Amplifies Adipogenesis of Human Bone Marrow-Derived Mesenchymal Stem Cells by Enhancing the Pro-Adipogenic Microenvironment. Tissue Eng. Part A 20, 966–981 (2014).

14. Lareu, R. R. et al. Collagen matrix deposition is dramatically enhanced in vitro when crowded with charged macromolecules: The biological relevance of the excluded volume effect. FEBS Lett. 581, 2709–2714 (2007).

15. Benny, P. & Raghunath, M. Making microenvironments: A look into incorporating macromolecular crowding into in vitro experiments, to generate biomimetic microenvironments which are capable of directing cell function for tissue engineering applications. J. Tissue Eng. 8, 1–8 (2017).

16. Benny, P., Badowski, C., Birgitte Lane, E. & Raghunath, M. Making More Matrix: Enhancing the Deposition of Dermal–Epidermal Junction Components In Vitro and Accelerating Organotypic Skin Culture Development, Using Macromolecular Crowding. Tissue Eng. Part A 21, 183–192 (2015).

17. Zeiger, A. S., Loe, F. C., Li, R., Raghunath, M. & van Vliet, K. J. Macromolecular Crowding Directs Extracellular Matrix Organization and Mesenchymal Stem Cell Behavior. PLoS One 7, 1–10 (2012).

18. Lee, M. H. et al. ECM microenvironment unlocks brown adipogenic potential of adult human bone marrow-derived MSCs. Sci. Rep. 6, 1–16 (2016).

19. Wong, C. W. et al. In Vitro Expansion of Keratinocytes on Human Dermal Fibroblast-Derived Matrix Retains Their Stem-Like Characteristics. Sci. Rep. 9, 1–17 (2019).

20. Ramalingam, R., Jiang, G., Larjava, H. & Häkkinen, L. Macromolecular crowding regulates matrix composition and gene expression in human gingival fibroblast cultures. Sci. Rep. 13, 1–20 (2023).

21. Michels, A. J. & Frei, B. Myths, artifacts, and fatal flaws: Identifying limitations and opportunities in vitamin C research. Nutrients 5, 5161–5192 (2013).

22. Yin, X. et al. Chemical Stability of Ascorbic Acid Integrated into Commercial Products: A Review on Bioactivity and Delivery Technology. Antioxidants 11, 1–20 (2022).

Download PDF

Version 1

posted

You are reading this older protocol version

Read the latest protocol version →

Inclusion of macromolecular crowders in three-dimensional human gingival fibroblast cultures

Status:

Version 1

Abstract

Figures

Introduction

Reagents

Equipment

Procedure

References

Associated Publications

Status:

Version 1

Privacy Policy

Terms of Service

Inclusion of macromolecular crowders in three-dimensional human gingival fibroblast cultures

Status:

Version 1

Abstract

Figures

Introduction

Reagents

Equipment

Procedure

References

Associated Publications

Status:

Version 1

Privacy Policy

Terms of Service

Manage Cookie Preferences