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Human Protein‐Based Porous Scaffolds as Platforms for Xeno‐Free 3D Cell Culture

Advanced Healthcare Materials

February 2022
11(12)

DOI:10.1002/adhm.202102383

Authors:

Sara Santos

University of Aveiro

Catarina De Almeida Custodio

University of Aveiro

João F. Mano

University of Aveiro

Extracellular matrix and protein‐based biomaterials emerge as attractive sources to produce scaffolds due to their great properties regarding biocompatibility and bioactivity. In addition, there are concerns regarding the use of animal‐derived supplements in cell culture not only due to the risk of transmission of xenogeneic contaminants and antigens but also due to ethical issues associated with collection methods. Herein, a novel human protein‐derived porous scaffold produced from platelet lysates (PL) as platform for xeno‐free 3D cell culture has been proposed. Human PL are chemically modified with methacryloyl groups (PLMA) to make them photocrosslinkable and used as precursor material to produce PLMA‐based sponges. The herein reported human‐based sponges have highly tunable morphology and mechanical properties, with an internal porous structure and Young's modulus dependent on the concentration of the polymer. Human adipose‐derived stem cells (hASCs) are cultured on top of PLMA sponges to validate their use for 3D cell culture in xeno‐free conditions. After 14 days hASCs remained viable, and results show that cells are able to proliferate during time even in the absence of animal‐derived supplementation. This study reveals for the first time that such scaffolds can be promising platforms for culture of human cells avoiding the use of any animal‐derived supplement.

Schematic representation of PL modification, PLMA sponges production and hASCs culture. PLMA was synthesized by reaction of PL with methacrylic anhydride. PLMA sponges were produced by a simple freeze‐drying technique and afterwards hASCs were cultured on top of PLMA sponges in medium with or without FBS supplementation.

…

PLMA‐based sponges characterization. a) CryoSEM representative images of PLMA scaffolds internal structure. Scale bar: 30 µm b) Pore size measurements analysis from cryoSEM images by ImageJ, represented by median, maximum and minimum values of the measured pore sizes. c) Young's modulus (kPa) of the studied scaffolds previously hydrated in PBS. d) Swelling ratio (%) of PLMA sponges after distinct immersion times in PBS at 37 °C. For all analysis PLMA10, PLMA15 and PLMA20 were used. Statistical analysis through one‐way ANOVA showed significant differences (*p < 0.05, ****p < 0.0001) between the analyzed groups. (n = 3).

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Live/Dead assay and DAPI/Phalloidin staining. Representative images of Live/Dead and DAPI/Phalloidin staining for hASCs cultured on top of PLMA15 sponges and SpongeCol at 3, 7, and 14 days of cell culture with and without animal‐derived serum supplement. DAPI/Phalloidin staining cross‐section representative images of hASCs cultured in PLMA sponges at 14 days. Scale bar: 100 µm.

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Cell proliferation and viability assessment. a) DNA and b) MTS results for hASCs after 3, 7, and 14 days of cell culture with and without animal‐derived serum supplement. Statistical analysis through two‐way ANOVA combined with Tukey's multiple comparisons test showed significant differences between the analyzed groups: *p < 0.05, **p < 0.01, ****p < 0.0001.

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PLMA growth factors quantification and protein release profile of PLMA‐based sponges. a) Quantification of PLMA growth factors by LEGENDplex Human Growth Factor Panel. b) Mass loss (%) of PLMA15 sponges. c) Total protein release quantification of PLMA15 sponges. d) Quantification of specific proteins and growth factors (Fibrinogen and VEGF) released by PLMA15 sponges. Protein release assays were performed with PLMA15 during 14 days.

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RESEARCH ARTICLE

www.advhealthmat.de

Human Protein-Based Porous Scaﬀolds as Platforms for

Xeno-Free 3D Cell Culture

Sara C. Santos, Catarina A. Custódio,* and João F. Mano*

Extracellular matrix and protein-based biomaterials emerge as attractive

sources to produce scaﬀolds due to their great properties regarding

biocompatibility and bioactivity. In addition, there are concerns regarding the

use of animal-derived supplements in cell culture not only due to the risk of

transmission of xenogeneic contaminants and antigens but also due to ethical

issues associated with collection methods. Herein, a novel human

protein-derived porous scaﬀold produced from platelet lysates (PL) as

platform for xeno-free 3D cell culture has been proposed. Human PL are

chemically modiﬁed with methacryloyl groups (PLMA) to make them

photocrosslinkable and used as precursor material to produce PLMA-based

sponges. The herein reported human-based sponges have highly tunable

morphology and mechanical properties, with an internal porous structure and

Young’s modulus dependent on the concentration of the polymer. Human

adipose-derived stem cells (hASCs) are cultured on top of PLMA sponges to

validate their use for 3D cell culture in xeno-free conditions. After 14 days

hASCs remained viable, and results show that cells are able to proliferate

during time even in the absence of animal-derived supplementation. This

study reveals for the ﬁrst time that such scaﬀolds can be promising platforms

for culture of human cells avoiding the use of any animal-derived supplement.

1. Introduction

The development of platforms for D cell culture remains a chal-

lenge in tissue engineering (TE). An ideal scaold requires spe-

ciﬁc biochemical and mechanical features close to those experi-

enced by the cells in their native tissues. Mechanical strength,

good porosity, biocompatibility, and good cell attachment are

important characteristics that should be taken into account

during scaold development process.[– ] In this sense, natu-

ral and synthetic macromolecules have been explored to pro-

duce scaolds for cell culture and TE. Despite all the advan-

tages found in synthetic-based scaolds regarding their mechan-

ical properties, they usually lack bioactivity and have limited

S. C. Santos, C. A. Custódio, J. F. Mano

Department of Chemistry

CICECO

University of Aveiro

Campus Universitário de Santiago, Aveiro 3810-193, Portugal

E-mail: catarinacustodio@ua.pt; jmano@ua.pt

The ORCID identiﬁcation number(s) for the author(s) of this article

can be found under https://doi.org/10.1002/adhm.202102383

DOI: 10.1002/adhm.202102383

biodegradability. Natural-based polymers

have emerged as promising materials due

to their great properties regarding bio-

compatibility, low immunogenicity, and

availability.[] However, they usually suer

from poor mechanical properties and sta-

bility in vitro.[] Therefore, strategies based

on chemical modiﬁcation of natural poly-

mers, physical crosslinking methods or pro-

duction of hybrid materials by conjugating

natural and synthetic materials have been

proposed.[] Extracellular matrix (ECM) and

protein-based biomaterials are particularly

appealing for cell culture and TE strate-

gies, since they are major components of

the cellular native microenvironment.[] In

this sense, the fabrication of porous scaf-

folds using collagen,[, ] gelatin,[, ] decel-

lularized ECM,[, ] albumin,[] keratin,[]

and silk ﬁbroin[, ] has been widely re-

ported.

Nowadays, there is a major concern re-

garding the use of animal-derived materials

and serum supplements in cell culture pro-

cedures due to the inherent risk of contami-

nation related with their non-human origin.

Besides that, there are also ethical issues related with collec-

tion methods of animal-derived products and potential limited

availability.[] Moreover, the use of animal-derived supplements

is associated with a risk of transmission of xenogeneic infectious

agents and immunization, making translation to clinical applica-

tions a more dicult process. Human-derived supplements such

as platelet lysates (PL) have been proposed as a replacement of

animal-derived supplements such as fetal bovine serum (FBS)

with particular interest for the development of cellular products

in clinical setting.[, ]

Many of the concerns with using animal-derived supplements

for cell culture extend to animal-derived materials for cell culture

as well. There is a need to ﬁnd alternatives to the current gold

standards for cell culture, including Matrigel and animal colla-

gen. Their batch-to-batch variability, and animal-derived nature

led to experimental uncertainty and a lack of reproducibility.

PL have been used for TE and regenerative medicine purposes

as an autologous source of growth factors and other bioactive

proteins involved in tissue healing process.[] By taking advan-

tage of the clotting properties of PL due to their richness in

ﬁbrinogen, scaolds have been developed by mixing PL with

calcium and/or thrombin that will trigger gels formation by the

conversion of ﬁbrinogen to ﬁbrin.[, ] However, PL-derived

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Cell culture is an important and necessary process in drug discovery, cancer research, as well as stem cell study. Most cells are currently cultured using two-dimensional (2D) methods but new and improved methods that implement three-dimensional (3D) cell culturing techniques suggest compelling evidence that much more advanced experiments can be performed yielding valuable insights. When performing 3D cell culture experiments, the cell environment can be manipulated to mimic that of a cell in vivo and provide more accurate data about cell-to-cell interactions, tumor characteristics, drug discovery, metabolic profiling, stem cell research, and other types of diseases. Scaffold based techniques such as hydrogel-based support, polymeric hard material-based support, hydrophilic glass fiber, and organoids are employed, and each provide their own advantages and applications. Likewise, there are also scaffold free techniques used such as hanging drop microplates, magnetic levitation, and spheroid microplates with ultra-low attachment coating. 3D cell culture has the potential to provide alternative ways to study organ behavior via the use of organoids and is expected to eventually bridge the gap between 2D cell culture and animal models. The present review compares 2D cell culture to 3D cell culture, provides the details surrounding the different 3D culture techniques, as well as focuses on the present and future applications of 3D cell culture.

Human Platelet Lysates‐Based Hydrogels: A Novel Personalized 3D Platform for Spheroid Invasion Assessment

Article

Full-text available

Feb 2020

Fundamental physiologic and pathologic phenomena such as wound healing and cancer metastasis are typically associated with the migration of cells through adjacent extracellular matrix. In recent years, advances in biomimetic materials have supported the progress in 3D cell culture and provided biomedical tools for the development of models to study spheroid invasiveness. Despite this, the exceptional biochemical and biomechanical properties of human‐derived materials are poorly explored. Human methacryloyl platelet lysates (PLMA)‐based hydrogels are herein proposed as reliable 3D platforms to sustain in vivo‐like cell invasion mechanisms. A systematic analysis of spheroid viability, size, and invasiveness is performed in three biomimetic materials: PLMA hydrogels at three different concentrations, poly(ethylene glycol) diacrylate, and Matrigel. Results demonstrate that PLMA hydrogels perfectly support the recapitulation of the tumor invasion behavior of cancer cell lines (MG‐63, SaOS‐2, and A549) and human bone‐marrow mesenchymal stem cell spheroids. The distinct invasiveness ability of each cell type is reflected in the PLMA hydrogels and, furthermore, different mechanical properties produce an altered invasive behavior. The herein presented human PLMA‐based hydrogels could represent an opportunity to develop accurate cell invasiveness models and open up new possibilities for humanized and personalized high‐throughput screening and validation of anticancer drugs. Human methacryloyl platelet lysates (PLMA)‐based hydrogels have been recently proposed as a new cost‐effective and biologically relevant 3D platform. PLMA hydrogels are demonstrated to support invasion mechanisms of different human cell spheroids allowing a distinct invasiveness ability depending on their mechanical properties, thus validating their potential to develop accurate cell invasiveness models.

Flexible, Water-Absorbing Silk Fibroin Biomaterial Sponges with Unique Pore Structure for Tissue Engineering

Article

Jan 2020

Silk fibroin (SF) scaffolds are widely used in tissue engineering due to their biocompatibility and slow biodegradability. However, the relatively stiff mechanical properties and low permeability of these systems can limit some applications. In this study, a new type of water-stable silk sponge (ASF-PEG-S) was obtained by inducing nanoparticle (50-300 nm diam.) formation in SF solution by autoclaving, followed by freeze-drying and rinsing the dry sponges with low molecular weight (400 Da) polyethylene glycol (PEG400) to induce SF β-sheet structure formation and thus stability in water. With further extraction, the SF nanoparticles embedded in the sponges were removed, leaving nano-pores in the walls of round-shaped micro-size pores. The unique pore structure resulted in enhanced permeability and flexibility of these ASF-PEG-S when compared to other types of SF sponges, especially with respect to commonly used methanol-annealed SF sponges. In addition, ASF-PEG-S absorbed water nearly 40 times more than the dry weight, while the methanol-annealed sponges absorbed half this amount. When human fibroblasts were seeded and cultured on the ASF-PEG-S versus traditional SF methanol processed sponges, improved cell encapsulation, distribution and consistency in growth were observed, suggesting utility in tissue engineering and tissue repair applications in the future.

Human Protein‐Based Porous Scaffolds as Platforms for Xeno‐Free 3D Cell Culture

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