Silk/Fibroin Microcarriers for Mesenchymal Stem Cell Delivery: Optimization of Cell Seeding by the Design of Experiment

Abstract

In this methodological paper, lyophilized fibroin-coated alginate microcarriers (LFAMs) proposed as mesenchymal stem cells (MSCs) delivery systems and optimal MSCs seeding conditions for cell adhesion rate and cell arrangement, was defined by a Design of Experiment (DoE) approach. Cells were co-incubated with microcarriers in a bioreactor for different time intervals and conditions: variable stirring speed, dynamic culture intermittent or continuous, and different volumes of cells-LFAMs loaded in the bioreactor. Intermittent dynamic culture resulted as the most determinant parameter; the volume of LFAMs/cells suspension and the speed used for the dynamic culture contributed as well, whereas time was a less influencing parameter. The optimized seeding conditions were: 98 min of incubation time, 12.3 RPM of speed, and 401.5 µL volume of cells-LFAMs suspension cultured with the intermittent dynamic condition. This DoE predicted protocol was then validated on both human Adipose-derived Stem Cells (hASCs) and human Bone Marrow Stem Cells (hBMSCs), revealing a good cell adhesion rate on the surface of the carriers. In conclusion, microcarriers can be used as cell delivery systems at the target site (by injection or arthroscopic technique), to maintain MSCs and their activity at the injured site for regenerative medicine.

Silk‐based microcarriers: current developments and future perspectives

“…In the reported works [28, 58, 74], the preparation of alginate cores was achieved by dropping alginate droplets in a calcium chloride water solution. Silk/alginate biomaterials were obtained after immersing the cores in a 1.5% w/v silk fibroin solution.…”

“…Silk/alginate biomaterials were obtained after immersing the cores in a 1.5% w/v silk fibroin solution. Conventionally, these composites are immersed or washed with 96% (v/v) ethanol to induce the conformational transition of silk fibroin from insoluble β‐sheet conformation to a water‐soluble structure [28, 58, 74]. The biological response of the resulting materials was evaluated after seeding with adipose‐derived stem/stromal cells (ASCs).…”

“…Although the use of bioreactors to perform cell seeding on MCs is a promising way to mimic and simulate the biological environment in order to obtain 3D constructs, studies of fibroin‐based MCs with dynamic cell culture are still scarce [60–74].…”

“…Perteghella et al [58] and Duchi et al [28] followed a conventional static cell seeding process of MSCs in the MCs, whereas, in the work of Perucca Orfei et al [74], cells were co‐incubated with MCs in a bioreactor for different time intervals and conditions. In order to make silk fibroin/alginate more stable, the MCs were freeze‐dried before optimising the seeding conditions.…”

Composite Central Face Design—An Approach to Achieve Efficient Alginate Microcarriers

“…The search of the optimal condition for each parameter, by varying one parameter while all others are fixed, is time-consuming, requires a lot of experiments, and the interaction effects between parameters can be missed, which sometimes is essential for better optimization of a process. To overcome these drawbacks, the Design of Experiments (DoE) can be used, a statistical tool that enables a systematic and simultaneous combination of parameters, which can influence the Ca-alginate micro-vehicles, in a faster and cost-efficient way when compared with the common random experiment approach [12,13].…”