Transforming eukaryotic cell culture with macromolecular crowding

doi:10.1016/j.tibs.2021.04.006

Trends in Biochemical Sciences

Volume 46, Issue 10, October 2021, Pages 805-811

https://doi.org/10.1016/j.tibs.2021.04.006 Get rights and content

Highlights

In eukaryotic cell culture, macromolecular crowding (MMC) enhances and accelerates extracellular matrix (ECM) deposition.
Negative charge and polydispersity are key modulators of enhanced and accelerated ECM deposition.
MMC augments dynamic cell-ECM reciprocity in cell culture.
We argue that MMC, by allowing the development of functional and truly 3D cell-assembled tissue surrogates, may be the missing link between developing and translating cell-based regenerative medicine and drug discovery concepts.

In multicellular organisms, the intracellular and extracellular spaces are considerably packed with a diverse range of macromolecular species. Yet, standard eukaryotic cell culture is performed in dilute, and deprived of macromolecules culture media, that barely imitate the density and complex macromolecular composition of tissues. Essentially, we drown cells in a sea of media and then expect them to perform physiologically. Herein, we argue the use of macromolecular crowding (MMC) in eukaryotic cell culture for regenerative medicine and drug discovery purposes.

Section snippets

The role of extracellular matrix in reparative medicine and drug discovery

Cell-based reparative medicine and drug discovery utilise the inherent capacity of cells, the building blocks of life, to create tissue-like surrogates in vitro [1]. The efficiency and safety of in vitro organogenesis has been repeatedly demonstrated in clinical settings for a diverse range of indications (e.g., cardiac [2], cartilage [3], cornea [4], and skin [5]). Similarly, in vitro cell-based disease models are far more economical than in vivo models and have demonstrated high-throughput

The theory of MMC in homogeneous systems

The intracellular and extracellular spaces are highly crowded/dense environments (e.g., ~400 g/l cytoplasm of Escherichia coli [22] and ~2000 g/l human cortical bone [23]). MMC, as opposed to macromolecular confinement, describes equilibria and kinetics of macromolecular reactions and/or interactions occurring in a highly volume-occupied or crowded solution, independently of the predominance of any single molecular species (i.e., high concentration) [24]. As a result of electrostatic repulsion,

The rationale of MMC in eukaryotic cell culture (heterogeneous systems)

To understand the function of MMC in cell culture, one should consider the biosynthesis and deposition of collagen, the most abundant ECM protein [44]. In vivo, the enzymatic conversion of the de novo synthesised water-soluble procollagen to water-insoluble collagen is rapid [45], as the dense and macromolecule-rich extracellular milieu restricts the diffusion of procollagen and N- and C- proteinases. Contrariwise, in the traditionally used, dilute, and deprived of macromolecules [the only

Advances of MMC in biomedicine and unanswered questions

In our opinion, current data unambiguously demonstrate that MMC has the potential to be the missing link in the accelerated development of ECM-rich cell-assembled tissue moduli in vitro for regenerative medicine (e.g., development of organotypic devices for cornea [52], tendon [63], and skin [64]), drug discovery (e.g., development of in vitro fibrosis [65] and tumour [66] pathophysiology models), and biomedicine (e.g., development of cell-derived matrices for effective cell expansion [67],

Concluding remarks

Irrefutably, we are still at the early stages of identifying the optimal crowding molecule/cocktail for maximum ECM deposition in the shortest period of time, whilst effectively controlling cell fate. Further, to rationally select a macromolecular crowder for a specific application (e.g., developing a functional reparative therapy, devising an accurate in vitro disease model, and improving a cell culture technology), we still need to develop multifactorial theories (and validate them through

Acknowledgements

This work has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme, grant number 866126 and the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie, grant number 676338. This publication has emanated from research conducted with the financial support of Science Foundation Ireland (SFI) under grant numbers 15/CDA/3629 and 19/FFP/6982 and SFI and European Regional

Declaration of interests

The authors declare no conflicts of interest.

Glossary

Anomalous diffusion: deviations from normal diffusion (i.e., diffusion is either faster or slower).
Cell culture media: dilute solutions of amino acids, vitamins, inorganic salts, carbohydrates, growth factors, and hormones that enable cell growth in vitro. The only macromolecule (>50 kDa) present in cell culture media is albumin when animal sera are used.
Diffusion: the random spreading of molecules/particles from regions of high concentration to regions of low concentration, eventually resulting in

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Macromolecular crowding in human tenocyte and skin fibroblast cultures: A comparative analysis
2024, Materials Today Bio
Although human tenocytes and dermal fibroblasts have shown promise in tendon engineering, no tissue engineered medicine has been developed due to the prolonged ex vivo time required to develop an implantable device. Considering that macromolecular crowding has the potential to substantially accelerate the development of functional tissue facsimiles, herein we compared human tenocyte and dermal fibroblast behaviour under standard and macromolecular crowding conditions to inform future studies in tendon engineering. Basic cell function analysis made apparent the innocuousness of macromolecular crowding for both cell types. Gene expression analysis of the without macromolecular crowding groups revealed expression of tendon related molecules in human dermal fibroblasts and tenocytes. Protein electrophoresis and immunocytochemistry analyses showed significantly increased and similar deposition of collagen fibres by macromolecular crowding in the two cell types. Proteomics analysis demonstrated great similarities between human tenocyte and dermal fibroblast cultures, as well as the induction of haemostatic, anti-microbial and tissue-protective proteins by macromolecular crowding in both cell populations. Collectively, these data rationalise the use of either human dermal fibroblasts or tenocytes in combination with macromolecular crowding in tendon engineering.
Carrageenan as a macromolecular crowding agent in human umbilical cord derived mesenchymal stromal cell culture
2023, International Journal of Biological Macromolecules
Cell sheet tissue engineering requires prolonged in vitro culture for the development of implantable devices. Unfortunately, lengthy in vitro culture is associated with cell phenotype loss and substantially higher cost of goods, which collectively hinder clinical translation and commercialisation of tissue engineered medicines. Although macromolecular crowding has been shown to enhance and accelerate extracellular matrix deposition, whilst maintaining cellular phenotype, the optimal macromolecular crowding agent still remains elusive. Herein, we evaluated the biophysical properties of seven different carrageenan molecules at five different concentrations and their effect on human umbilical cord-derived mesenchymal stromal cell morphology, viability, metabolic activity, proliferation, extracellular matrix deposition and surface marker expression. All types of carrageenan (CR) assessed demonstrated a hydrodynamic radius increase as a function of increasing concentration; high polydispersity; and negative charge. Two iota CRs were excluded from further analysis due to poor solubility in cell culture. Among the remaining five carrageenans, the lambda medium viscosity type at concentrations of 10 and 50 μg/ml did not affect cell morphology, viability, metabolic activity, proliferation and expression of surface markers and significantly increased the deposition of collagen types I, III and IV, fibronectin and laminin. Our data highlight the potential of lambda medium viscosity carrageenan as a macromolecular crowding agent for the accelerated development of functional tissue engineered medicines.
Macromolecular crowding in equine bone marrow mesenchymal stromal cell cultures using single and double hyaluronic acid macromolecules
2023, Acta Biomaterialia
Macromolecular crowding (MMC) enhances and accelerates extracellular matrix (ECM) deposition in eukaryotic cell culture. Single hyaluronic acid (HA) molecules have not induced a notable increase in the amount and rate of deposited ECM. Thus, herein we assessed the physicochemical properties and biological consequences in equine bone marrow mesenchymal stromal cell cultures of single and mixed HA molecules and correlated them to the most widely used MMC agents, the Ficoll^Ⓡ cocktail (FC) and carrageenan (CR). Dynamic light scattering analysis revealed that all HA cocktails had significantly higher hydrodynamic radius than the FC and CR; the FC and the 0.5 mg/ml 100 kDa and 500 kDa single HA molecules had the highest charge; and, in general, all molecules had high polydispersity index. Biological analyses revealed that none of the MMC agents affected cell morphology and basic cell functions; in general, CR outperformed all other macromolecules in collagen type I and V deposition; FC, the individual HA molecules and the HA cocktails outperformed CR in collagen type III deposition; FC outperformed CR and the individual HA molecules and the HA cocktails outperformed their constituent HA molecules in collagen type IV deposition; FC and certain HA cocktails outperformed CR and constituent HA molecules in collagen type VI deposition; and all individual HA molecules outperformed FC and CR and the HA cocktails outperformed their constituent HA molecules in laminin deposition. With respect to tri-lineage analysis, CR and HA enhanced chondrogenesis and osteogenesis, whilst FC enhanced adipogenesis. This work opens new avenues in mixed MMC in eukaryotic cell culture.
Mixed macromolecular crowding (MMC) in eukaryotic cell culture is still under-investigated. Herein, single and double hyaluronic acid (HA) macromolecules, along with the traditional MMC agents Ficoll^Ⓡ cocktail (FC) and carrageenan (CR), were used as MMC agents in equine mesenchymal stromal cell cultures. Biological analysis showed that none of the MMC agents affected cell morphology and basic cell functions. Protein deposition analysis made apparent that CR outperformed all other macromolecules in collagen type I and collagen type V deposition, whilst FC, the individual HA macromolecules and the HA cocktails outperformed CR in collagen type III deposition. Tri-lineage analysis revealed that CR and HA enhanced chondrogenesis and osteogenesis, whilst FC enhanced adipogenesis. These data illustrate that MMC agents are not inert macromolecules.
Physicochemical cues are not potent regulators of human dermal fibroblast trans-differentiation
2023, Biomaterials and Biosystems
Due to their inherent plasticity, dermal fibroblasts hold great promise in regenerative medicine. Although biological signals have been well-established as potent regulators of dermal fibroblast function, it is still unclear whether physiochemical cues can induce dermal fibroblast trans-differentiation. Herein, we evaluated the combined effect of surface topography, substrate rigidity, collagen type I coating and macromolecular crowding in human dermal fibroblast cultures. Our data indicate that tissue culture plastic and collagen type I coating increased cell proliferation and metabolic activity. None of the assessed in vitro microenvironment modulators affected cell viability. Anisotropic surface topography induced bidirectional cell morphology, especially on more rigid (1,000 kPa and 130 kPa) substrates. Macromolecular crowding increased various collagen types, but not fibronectin, deposition. Macromolecular crowding induced globular extracellular matrix deposition, independently of the properties of the substrate. At day 14 (longest time point assessed), macromolecular crowding downregulated tenascin C (in 9 out of the 14 groups), aggrecan (in 13 out of the 14 groups), osteonectin (in 13 out of the 14 groups), and collagen type I (in all groups). Overall, our data suggest that physicochemical cues (such surface topography, substrate rigidity, collagen coating and macromolecular crowding) are not as potent as biological signals in inducing dermal fibroblast trans-differentiation.
Scaffolding design and structure/function
2023, Multiscale Cell-Biomaterials Interplay in Musculoskeletal Tissue Engineering and Regenerative Medicine
Electrospinning is continuously gaining pace in regenerative medicine, as it allows for the development of implantable devices that closely resemble the structural features and mechanical properties of tissues. Indeed, advances in engineering have enabled the development of electrospun scaffolds with the ability to deliver in localized and sustained manner in a diverse range of bioactive therapeutic molecules. Naïve, through architectural features and/or mechanical properties, and functionalized, through biochemical and/or biological signals, electrospun scaffolds have also been shown to both maintain permanently differentiated cell phenotype and direct mesenchymal stromal cell lineage commitment. Considering that tendon injuries and pathophysiologies constitute an unmet clinical need, herein, we summarize recent advances of naïve, molecular, and cellular electrospinning in the tendon engineering context.
The synergistic effect of physicochemical in vitro microenvironment modulators in human bone marrow stem cell cultures
2023, Biomaterials Advances
Citation Excerpt :
It is worth noting that carrageenan has been used extensively as MMC agent with no negative effects in a diverse range of cell types (e.g. hBMSCs [33,34,48], human adipose derived stem cells [38], equine adipose derived stem cells [39], human dermal fibroblasts [37,80], human chondrocytes [81], human corneal fibroblasts [40], human tenocytes [35,82]). The most noticeable feature in this set of experiments is that MMC at all time points considerably increased collagen types I, III, IV, V and VI deposition, thanks to its well-established excluded volume effect that enhances the enzymatic conversion of procollagen to collagen [27–29]. Fibronectin deposition on the other hand was not increased, as fibronectin is not enzymatically processed [83].
Modern bioengineering utilises biomimetic cell culture approaches to control cell fate during in vitro expansion. In this spirit, herein we assessed the influence of bidirectional surface topography, substrate rigidity, collagen type I coating and macromolecular crowding (MMC) in human bone marrow stem cell cultures. In the absence of MMC, surface topography was a strong modulator of cell morphology. MMC significantly increased extracellular matrix deposition, albeit in a globular manner, independently of the surface topography, substrate rigidity and collagen type I coating. Collagen type I coating significantly increased cell metabolic activity and none of the assessed parameters affected cell viability. At day 14, in the absence of MMC, none of the assessed genes was affected by surface topography, substrate rigidity and collagen type I coating, whilst in the presence of MMC, in general, collagen type I α1 chain, tenascin C, osteonectin, bone sialoprotein, aggrecan, cartilage oligomeric protein and runt-related transcription factor were downregulated. Interestingly, in the presence of the MMC, the 1000 kPa grooved substrate without collagen type I coating upregulated aggrecan, cartilage oligomeric protein, scleraxis homolog A, tenomodulin and thrombospondin 4, indicative of tenogenic differentiation. This study further supports the notion for multifactorial bioengineering to control cell fate in culture.

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Trends in Biochemical Sciences

OpinionTransforming eukaryotic cell culture with macromolecular crowding

Highlights

Section snippets

The role of extracellular matrix in reparative medicine and drug discovery

The theory of MMC in homogeneous systems

The rationale of MMC in eukaryotic cell culture (heterogeneous systems)

Advances of MMC in biomedicine and unanswered questions

Concluding remarks

Acknowledgements

Declaration of interests

Glossary

Cytotherapy

Trends Biotechnol.

Dev. Cell

Trends Cell Biol.

Cytotherapy

Cell. Signal.

J. Orthop. Res.

Growth Hormon. IGF Res.

J. Mol. Biol.

Trends Biochem. Sci.

Int. J. Biol. Macromol.

FEBS Lett.

J. Biol. Chem.

Acta Biomater.

Int. J. Biol. Macromol.

Anal. Biochem.

FEBS Lett.

Acta Biomater.

Biomaterials

Biomaterials

Biochem. Biophys. Res. Commun.

Trends Biotechnol.

Acta Biomater.

Biomater. Biosyst.

Adv. Drug Deliv. Rev.

Biochim. Biophys. Acta Gen. Subj.

Mater. Sci. Eng. C Mater. Biol. Appl.

Harnessing the secreted extracellular matrix to engineer tissues

Nat. Biomed. Eng.

Phase I clinical trial of autologous stem cell-sheet transplantation therapy for treating cardiomyopathy

J. Am. Heart Assoc.

Combined surgery and chondrocyte cell-sheet transplantation improves clinical and structural outcomes in knee osteoarthritis

NPJ Regen. Med.

Phase 1 clinical study of an embryonic stem cell-derived retinal pigment epithelium patch in age-related macular degeneration

Nat. Biotechnol.

Regeneration of the entire human epidermis using transgenic stem cells

Nature

c-myc regulates the sensitivity of breast cancer cells to palbociclib via c-myc/miR-29b-3p/CDK6 axis

Cell Death Dis.

APOE4 exacerbates synapse loss and neurodegeneration in Alzheimer's disease patient iPSC-derived cerebral organoids

Nat. Commun.

AdipoR1/AdipoR2 dual agonist recovers nonalcoholic steatohepatitis and related fibrosis via endoplasmic reticulum–mitochondria axis

Nat. Commun.

Theranostic drug test incorporating the bone-marrow microenvironment can predict the clinical response of acute myeloid leukaemia to chemotherapy

Br. J. Haematol.

Human tissue-engineered blood vessels for adult arterial revascularization

Nat. Med.

High-resolution tomographic analysis of in vitro 3D glioblastoma tumor model under long-term drug treatment

Sci. Adv.

Dynamic reciprocity revisited: a continuous, bidirectional flow of information between cells and the extracellular matrix regulates mammary epithelial cell function

Biochem. Cell Biol.

Low oxygen tension stimulates collagen synthesis and COL1A1 transcription through the action of TGF-beta1

J. Cell. Physiol.

Simulation of exposure and SAR estimation for adult and child heads exposed to radiofrequency energy from portable communication devices

Radiat. Res.

Beyond the excluded volume effects: mechanistic complexity of the crowded milieu

Molecules

Analysis of the size dependence of macromolecular crowding shows that smaller is better

Proc. Natl. Acad. Sci. U. S. A.

Macromolecular crowding acts as a physical regulator of intracellular transport

Nat. Phys.

Crowding and hydrodynamic interactions likely dominate in vivo macromolecular motion

Proc. Natl. Acad. Sci. U. S. A.

Opinion
Transforming eukaryotic cell culture with macromolecular crowding