Based on the results of fluorescence detection, we can infer that miR-148a-5p has been almost entirely transfected into the exosomes. A portion of the miRNAs in the miR-455-3p group were transfected into the exosomes, thus obtaining exosomes loaded with these two miRNAs.

Measuring the OD value to determine whether miRNA has been transferred into exosomes. The fluorescence report is as follows:

miR-455-3p:

miR-148a-5p:

The exosomes loaded with miRNA provided by the company are as follows

Exosomes loaded with miRNA:

After being cultured in complete medium, resuscitated hepatic stellate cells (HSCs) exhibit the characteristics of a significant increase in density and a morphological transition from the quiescent state to the activated state under an optical microscope. At this stage, the cells have passed the initial adaptation phase and entered the late logarithmic growth phase; in terms of density, they can cover 70%-90% of the bottom area of the culture dish, with a slightly sparse distribution at the edges and a uniform distribution in the central area. Morphologically, the cells no longer have the round or oval shape (in the state of incomplete adherence or just adherent) observed in the early resuscitation stage, but transform into a spindle-shaped, polygonal, or long spindle-shaped morphology similar to myofibroblasts. Their cell bodies are significantly stretched with increased volume and abundant cytoplasm; the cell processes increase in number and length, and interconnect with the processes of adjacent cells to form a network structure.

Changes in Cell Density During Cell Culture (Taking Cell Resuscitation to the First Passage as an Example)

Day1

Day2

Day3

Day4

Day5

Day6

Before induction, quiescent hepatic stellate cells (HSCs) adhere to the substrate and grow. They exhibit a typical "spindle-shaped" or "polygonal" morphology, with small cell bodies. Additionally, the cells extend multiple long, slender cytoplasmic protrusions that interconnect to form a network-like structure.After induction with TGF-β, cell spacing decreases, and cell stacking even occurs in some regions. Meanwhile, the cell morphology transitions from "spindle-shaped" to "fusiform" (similar to fibroblasts). The cell bodies increase significantly in size, and the cytoplasmic staining deepens—this is due to increased expression of cytoskeletal proteins such as α-smooth muscle actin (α-SMA) in the cytoplasm. Furthermore, the original long, slender protrusions shorten and thicken, and the intercellular connections decrease.

LX-2 cells in normal condition

LX-2 cells induced for 24 hours

LX-2 cells induced for 48 hours

LX-2 cells induced for 72 hours