Scalable Production of Standardized Extracellular Vesicles, Extracellular Vesicle Preparations and Uses Thereof

A scalable method for producing an enriched population of negatively charged extracellular vesicles, said method comprising:

• (a) culturing a population of undifferentiated human cells under appropriate conditions suitable for inducing differentiation of said cells to mesenchymal stem cells;
• (b) culturing said induced population of human cells for between about 6 hours and about 48 hours in a supplemented, protein-free culture medium that prevents cell aggregation of cells synthesizing TSG-6, to produce a conditioned culture medium;
• (c) collecting the conditioned media and removing cellular debris from said conditioned culture medium;
• (d) passing the conditioned culture medium over an anion exchange column to provide a population of extracellular vesicles having a negative surface charge (n-EVs) attached to said column;
• (e) removing the population of n-EVs from the column;
• and
• (f) selecting from said population of n-EVs a subset of n-EVs having activity for suppressing neuroinflammation and having a surface epitope that is absent CD9 (CD9).

A method for reducing brain inflammation comprising:

• (a) administering a pharmaceutically acceptable preparation comprising an enriched preparation of negatively charged n-EVs and absent a CD9 epitope (CD9−) to an animal at risk of having or having inflammation attendant traumatic brain injury;
• and
• (b) observing a reduction in brain inflammation in the animal,
• wherein a reduction in brain inflammation is correlated with a return to consciousness, improved mobility, or other physically manifested improvement.

A method for improving pattern separation function and spatial learning ability in an animal subsequent a traumatic brain injury (TBI) comprising:

• (a) providing a base-line separation function and spatial learning ability of an animal having had a traumatic brain injury (TBI) to provide a base-line assessment measurement;
• (b) administering a pharmaceutically acceptable preparation comprising an enriched preparation of negatively charged n-EVs that are absent a CD9 epitope (CD9−) to an animal at risk of having or having inflammation attendant traumatic brain injury to provide a treated animal;
• (c) assessing the treated animal for separation function and spatial learning ability to provide post-treatment assessment measurements, and comparing the post-treatment assessment measurements to the base-line separation function and spatial learning assessment measurements;
• and
• (d) providing an animal having improved pattern separation function and spatial learning ability where the post-treatment assessment measurements are improved compared to the base-line assessment measurements.

An enriched population of pharmacologically active n-EVs that have a CD9− surface epitope, prepared by a method comprising the steps of:

• (a) culturing a population of undifferentiated human cells under appropriate conditions suitable for inducing differentiation of said cells to mesenchymal stem cells;
• (b) culturing said induced population of human cells for between about 6 hours and about 48 hours in a supplemented, protein-free culture medium that prevents cell aggregation of cells synthesizing TSG-6, to produce a conditioned culture medium;
• (c) collecting the conditioned media and removing cellular debris from said conditioned culture medium;
• (d) passing the conditioned culture medium over an anion exchange column to provide a population of extracellular vesicles having a negative surface charge (n-EVs) attached to said column;
• (e) removing the population of n-EVs from the column;
• and
• (f) selecting from said population of n-EVs a subset of n-EVs having activity for suppressing neuroinflammation and having a surface epitope that is absent CD9 (CD9−) to provide an enriched population of pharmacologically active n-EVs having a CD9− surface epitope.

A method for producing an enriched preparation of negatively charged extracellular vesicles (n-EVs) having brain anti-inflammatory pharmacological activity in vivo, comprising:

• (a) culturing a population of human mesenchymal stem cells for between about 6 hours and about 48 hours in a supplemented, protein-free culture medium that prevents cell aggregation of cells synthesizing TSG-6, to produce a conditioned culture medium;
• (b) collecting the conditioned media and removing cellular debris from said conditioned culture medium;
• (c) passing the conditioned culture medium over an anion exchange column to provide a population of extracellular vesicles having a negative surface charge (n-EVs) attached to said column;
• (d) removing the population of n-EVs from the column in an eluate from the column;
• and
• (e) selecting from said population of n-EVs a subset of n-EVs having activity for suppressing brain tissue neuroinflammation and having a surface epitope that is absent CD9 (CD9−) to provide an enriched population of pharmacologically active n-EVs having a CD9− surface epitope.

A method for treating concussion in a human subject comprising:

• (a) identifying a person who has experienced a concussion;
• (b) administering a pharmaceutically acceptable preparation comprising an enriched preparation of negatively charged, n-EVs that are absent a CD9 epitope (CD9−) to the person identified to have had a concussion;
• and
• (c) assessing the response of the person to the administered preparation,
• wherein a positive response to the administered preparation is identified in the treated person that evidences a state of regained consciousness.